Mag1 nucleic acid molecules and their uses

ABSTRACT

The methods and compositions of the present invention find use in impacting microbial pathogens and in enhancing disease resistance to pathogens, particularly by plants. The compositions of the invention include polypeptides that possess antimicrobial properties, particularly fungicidal properties, and the encoding nucleic acid molecules. The polypeptides of the invention are isolated from the hemolymph and fat bodies of insect larvae induced by injection of plant pathogenic fungi. Further provided are plant cells, plants, and seed thereof, transformed with the nucleic acid molecules of the invention so as to confer disease resistance on the plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/125,258, filed Apr. 18, 2002, which claims the benefit of U.S.Provisional Application No. 60/285,355, filed Apr. 20, 2001, both ofwhich applications are hereby incorporated herein in their entirety byreference.

FIELD OF THE INVENTION

The invention relates to plant disease resistance, particularlyresistance to fungal pathogens. More specifically the present inventionrelates to the use of naturally occurring antimicrobial polypeptidesisolated from insects induced with plant pathogens.

BACKGROUND OF THE INVENTION

Multicellular organisms produce a battery of antimicrobial peptides andproteins to defend themselves against microbial attack or injury. Manyof these induced peptides and proteins possess broad antimicrobialactivity against Gram-positive and/or Gram-negative bacteria (Boman, H.G. (1995) Annu. Rev. Immunol. 13:61-92). This defense system, called“innate immunity,” may represent a chemical barrier that organismsdeploy to stop dangerous microbes at their point of contact.

The peptides and proteins produced in response to microbial attack tendto work very differently from conventional antibiotics. Antibiotics workto block a crucial protein in an invading microbe. The mode of action ofthe antimicrobial defensive proteins varies. In some instances, theypunch holes in a microbe's membranes and disrupt internal signaling ofthe microbe. In other instances, they may act to increase the host cellimmune activity.

Several antimicrobial peptides have been isolated and their structurespartially characterized. The defensins, one type of the antimicrobialpeptides, are cysteine-rich peptides. Defensins have been isolated frominsects and mammals. Insect defensins are 34-43 amino acid peptides withthree disulfide bridges. They are produced by the insect fat body(Hoffmann et al. (1992) Immunol. Today 13:411-15). They have been shownto disrupt the permeability of the cytoplasmic membrane of Micrococcusluteus, resulting from the formation of voltage-dependent ion channelsin the cytoplasmic membrane (Cociancich et al. (1993) J. Biol. Chem.268:19239-19245).

Thionins are another group of small cysteine-rich antimicrobialpeptides. Thionins are thought to play a role in the protection ofplants against microbial infection. They are found in the seedendosperm, stems, roots, and in etiolated or pathogen stressed leaves ofmany plant species (Bohlmann et al. (1991) Annu. Rev. Plant Physiol.Plant Mol. Biol. 42:227-240). Thionins display toxicity to bacteria,fungi, yeasts, and even various mammalian cell types.

Disease in plants has many causes including fungi, viruses, bacteria,and nematodes. Phytopathogenic fungi have resulted in significant annualcrop yield losses as well as devastating epidemics. Additionally, plantdisease outbreaks have resulted in catastrophic crop failures that havetriggered famines and caused major social change.

Molecular methods of crop protection not only have the potential toimplement novel mechanisms for disease resistance, but can also beimplemented more quickly than traditional breeding methods. Accordingly,molecular methods are needed to supplement traditional breeding methodsto protect plants from pathogen attack.

Plant pathogenic fungi attack all of the approximately 300,000 speciesof flowering plants, but a single plant species can be host to only afew fungal species, and most fungi usually have a limited host range. Itis for this reason that the best general strategy to date forcontrolling plant fungal disease has been to use resistant cultivarsselected or developed by plant breeders. Unfortunately, even with theuse of resistant cultivars, the potential for serious crop diseaseepidemics persists today, as evidenced by outbreaks of Victoria oat andsouthern corn leaf blight.

Accordingly, molecular methods utilizing the resistance mechanisms ofnaturally occurring plant insect pests to enhance plant diseaseresistance to microbes, particularly pathogenic fungi, are desirable.

SUMMARY OF THE INVENTION

Compositions and methods for increasing resistance to pathogens areprovided. The compositions comprise antipathogenic peptides or defensiveagents that are induced in insects by contacting the insect with apathogen of interest. The compositions include polypeptides that possessantimicrobial properties, particularly fungicidal properties, and thenucleic acid molecules that encode such polypeptides. The methods andcompositions of the present invention find use in impacting plantmicrobial pathogens and in enhancing plant disease resistance tomicrobial pathogens.

Expression cassettes comprising the nucleic acid molecules encoding thedefensive agents, vector sequences and host cells for the expression ofthe polypeptides, and antibodies to the polypeptides are also provided.The compositions of the invention further provide plant cells, plants,and seed thereof, transformed with the nucleic acid molecules of theinvention. The transgenic plants of the present invention aretransformed with a nucleotide sequence of the invention and exhibitincreased antimicrobial disease resistance, particularly fungal diseaseresistance that will lessen the need for artificial agriculturalchemicals to protect field crops and increase crop yield.

The methods of the invention involve stably transforming a plant with atleast one expression cassette comprising at least one nucleotidesequence of the invention operably linked with a promoter capable ofdriving expression of the nucleotide sequence in the plant or plantcell. It is recognized that a variety of promoters will be useful in theinvention, the choice of which will depend in part upon the desiredtissue localization and the level of expression of the disclosednucleotide sequences and corresponding polypeptides. It is recognizedthat the levels of expression of the defensive agents in the plant cellcan be controlled so as to achieve optimal disease resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence alignment of precursor Mag1 polypeptide (SEQID NO:2) with the class of immune proteins known as attacins. Theprecursor Mag1 polypeptide has 78% sequence similarity with attacin E/Fprecursor polypeptide (SEQ ID NO:19, Accession No: P01513). Theremaining sequences are: Attacin A precursor polypeptide (SEQ ID NO:17,Accession No: P50725); Attacin B precursor polypeptide (SEQ ID NO:18,Accession No: P01512); and the attacin precursor polypeptide known asNuecin (SEQ ID NO:20, Accession No: Q26431).

FIG. 2. Amino acid sequence alignment of precursor Mag1 polypeptide (SEQID NO:2) with homologous polypeptide sequences of the invention encodedby cDNAs isolated from pathogen induced Manduca sexta libraries (SEQ IDNOS:4, 6, 8, and 10).

FIG. 3. The N-terminal amino acid sequences for the four Mag1polypeptide Lys-C digestion fragments (SEQ ID NO:96, 97, 98, and 99).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for enhancingplant disease resistance to plant pathogens, particularly fungalpathogens. The compositions of the invention include polypeptides andpeptides that possess antimicrobial activity, particularly fungicidalactivity. Such peptides or polypeptides are collectively referred to as“defensive agents” herein. Nucleic acid molecules encoding suchdefensive agents, as well as plants transformed with the nucleic acidmolecules, are also included.

The invention is drawn to compositions and methods for inducingresistance in a plant to plant pests. The defensive agents compriseinsect derived nucleotide and polypeptide sequences. Accordingly, thecompositions and methods are also useful in protecting plants againstfungal pathogens, viruses, nematodes, and the like.

Compositions for controlling plant pathogenic agents, particularly plantpathogenic microbial agents, more particularly plant pathogenic fungalagents are provided. Specific compositions provided includeinsect-derived antimicrobial polypeptides and the nucleic acid moleculesencoding such polypeptides. Plants, plant cells, plant tissues and seedsthereof transformed with the nucleotide sequences of the invention areprovided. Additionally, the compositions of the invention can be used informulations for their disease resistance activities.

The present invention provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequences shownin SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 22, 23, 25, 26, 28, 29, 31,32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58,59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85,86, 88, 89, 91, 92, 94, 95, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, or 127. Further provided are polypeptides having anamino acid sequence encoded by a nucleic acid molecule described herein,for example, those set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 21,24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75,78, 81, 84, 87, 90, 93, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, or 126, and fragments and variants thereof.

Methods are provided for the expression of these sequences in a hostplant to confer enhanced disease resistance of the host plant to plantpathogens, particularly plant fungal pathogens. The methods of theinvention involve stably transforming a plant with at least oneexpression cassette comprising at least one nucleotide sequence of theinvention operably linked with a promoter capable of driving expressionof the nucleotide sequence in the plant cell. It is recognized that avariety of promoters will be useful in the invention, the choice ofwhich will depend in part upon the desired level and desired tissuelocalization of expression of the disclosed nucleotide sequences. It isrecognized that the levels and tissue location of expression can becontrolled to modulate the levels of the antimicrobial polypeptides inthe plant cell to optimize plant disease resistance to a particularpathogen.

By “plant pathogen” or “plant pest” is intended any microorganism thatcan cause harm to a plant, such as by inhibiting or slowing the growthof a plant, by damaging the tissues of a plant, by weakening the immunesystem of a plant or the resistance of a plant to abiotic stresses,and/or by causing the premature death of the plant, etc. Plant pathogensand plant pests include microbes such as fungi, viruses, bacteria, andnematodes.

By “disease resistance” or “pathogen resistance” is intended that theplants avoid the disease symptoms which are the outcome of plantpathogen interactions. That is, pathogens are prevented from causingplant diseases and the associated disease symptoms, or alternatively,the disease symptoms caused by the pathogen are minimized or lessened.The methods of the invention can be utilized to protect plants fromdisease, particularly those diseases that are caused by plant fungalpathogens.

An “antimicrobial agent,” a “pesticidal agent,” a “defensive agent,”and/or a “fungicidal agent” will act similarly to suppress, control,and/or kill the invading pathogen.

A defensive agent will possess defensive activity. By “defensiveactivity” is intended an antipathogenic, antimicrobial, or antifungalactivity.

By “antipathogenic compositions” is intended that the compositions ofthe invention have activity against pathogens including fungi,microorganisms, viruses, and nematodes and thus are capable ofsuppressing, controlling, and/or killing the invading pathogenicorganism. An antipathogenic composition of the invention will reduce thedisease symptoms resulting from microbial pathogen challenge by at leastabout 5% to about 50%, at least about 10% to about 60%, at least about30% to about 70%, at least about 40% to about 80%, or at least about 50%to about 90% or greater. Hence, the methods of the invention can beutilized to protect organisms, particularly plants, from disease,particularly those diseases that are caused by invading pathogens.

Assays that measure antipathogenic activity are commonly known in theart, as are methods to quantify disease resistance in plants followingpathogen infection. See, for example, U.S. Pat. No. 5,614,395, hereinincorporated by reference. Such techniques include measuring over timethe average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. For example, a plant eitherexpressing an antipathogenic polypeptide or having an antipathogeniccomposition applied to its surface shows a decrease in tissue necrosis(i.e., lesion diameter) or a decrease in plant death following pathogenchallenge when compared to a control plant that was not exposed to theantipathogenic composition. Alternatively, antipathogenic activity canbe measured by a decrease in pathogen biomass. For example, a plantexpressing an antipathogenic polypeptide or exposed to an antipathogeniccomposition is challenged with a pathogen of interest. Over time, tissuesamples from the pathogen-inoculated tissues are obtained and RNA isextracted. The percent of a specific pathogen RNA transcript relative tothe level of a plant specific transcript allows the level of pathogenbiomass to be determined. See, for example, Thomma et al. (1998) PlantBiology 95:15107-15111, herein incorporated by reference.

Furthermore, in vitro fungicidal assays include, for example, theaddition of varying concentrations of the fungicidal composition topaper disks and placing the disks on agar containing a suspension of thepathogen of interest. Following incubation, clear inhibition zonesdevelop around the discs that contain an effective concentration of thefungicidal polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892,herein incorporated by reference). Additional methods are used in theart to measure the in vitro fungicidal properties of a composition (Huet al. (1997) Plant Mol. Biol. 34:949-959; Cammue et al. (1992) J. Biol.Chem. 267:2228-2233; and Thevissen et al. (1996) J. Biol Chem.271:15018-15025, all of which are herein incorporated by reference).

Pathogens of the invention include but are not limited to viruses orviroids, bacteria, insects, nematodes, fungi, and the like. Virusesinclude any plant virus, for example, tobacco or cucumber mosaic virus,ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specificfungal and viral pathogens for the major crops include: Soybeans:Phytophthora megasperma f.sp. glycinea, Macrophomina phaseolina,Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthephaseolorum var. caulivora, Sclerotium roltsii, Cercospora kikuchii,Cercospora sojina, Peronospora manshurica, Colletotrichum dematium(Colletotichum truncatum), Corynespora cassiicola, Septoria glycines,Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v.glycinea, Xanthomonas campestris p.v. phaseoli, Microsphaera diffusa,Fusarium semitectum, Phialophora gregata, Soybean mosaic virus,Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus,Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythiumdebaryanum, Tomato spotted wilt virus, Heterodera glycines Fusariumsolani; Canola: Albugo candida, Alternaria brassicae, Leptosphaeriamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Alternaria alternata; Alfalfa: Clavibacter michiganensis subsp.insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens,Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma,Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercosporamedicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis,Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,Stemphylium herbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringaep.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata,Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum,Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporiumgramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici,Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici,Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum,Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides,Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var.tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum,Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus,Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat SpindleStreak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletiatritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctoniasolani, Pythium arrhenomanes, Pythium gramicola, Pythium aphanidermatum,High Plains Virus, European wheat striate virus; Sunflower: Plasmophorahalstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi,Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytiscinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphecichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer,Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum p.v.carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugotragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwiniastewartii, Fusarium verticilloides, Fusarium moniliforme, Gibberellazeae (Fusarium graminearum), Stenocarpella maydis (Diplodia maydis),Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythiumsplendens, Pythium ultimum, Pythium aphanidermatum, Aspergillusflavus,Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporiumcarbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II& III, Helminthosporium pedicellatum, Physoderma maydis, Phyllostictamaydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Pucciniasorghi, Puccinia polysora, Macrophomina phaseolina, Penicilliumoxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata,Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganensesubsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B,Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Clavicepssorghi, Pseudoinonas avenae, Erwinia chrysanthemi pv. zea, Erwiniacarotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthoramacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelothecareiliana, Physopella zeae, Cephalosporium maydis, Cephalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum,Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi,Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v.syringae, Xanthomonas campestris p.v. holcicola, Pseudomonasandropogonis, Puccinia purpurea, Macrophomina phaseolina, Periconiacircinata, Fusarium moniliforme, Alternaria alternata, Bipolarissorghicola, Helminthosporium sorghicola, Curvularia lunata, Phomainsidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulisporasorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisoriumreilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisoriumsorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Clavicepssorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthoramacrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis,Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,Pythium arrhenomanes, Pythium graminicola; Rice: Magnaporthe grisea,Rhizoctonia solani, etc.

The specific defensive agents of the invention have been demonstrated tohave antipathogenic activity against particular pathogens. It isrecognized that they may demonstrate activity against other pathogens,particularly other fungal pathogens. Some may even exhibitbroad-spectrum antipathogenic activity. It is recognized that whileantifungal polypeptides may demonstrate activity against a particularpest, such defensive agents may have activity against numerous fungalpathogens, as well as other plant pests. Thus, a plant transformed witha particular defensive agent of the invention may demonstratebroad-spectrum resistance.

In one embodiment of the invention, defensive agents are isolated fromthe hemolymph of insect larvae induced by injection of a plantpathogenic fungi. The antimicrobial polypeptides induced can be placedinto at least four groups according to their amino acid sequencehomology to known classes of proteins. These four groups consist of theattacin, lebocin, and serine protease inhibitor classes of proteins, anda group that does not demonstrate substantial homology to knownproteins. The defensive agents enhance disease resistance to fungalpathogens, Magnathorpa grisea (M. grisea), Rhizoctonia solani (R.solani), and Fusarium verticilloides (F. verticilloides). Specifically,the polypeptides of the invention were identified from the hemolymph ofinsect larvae induced by injection of the plant pathogenic fungi, M.grisea, R. solani, or F. verticilloides.

The compositions of the invention comprise M. sexta (tobacco hornworm),Heliothis virescens (tobacco budworm), Ostrinia nubilalis (Europeancomborer), Peregrinus maidis (complant hopper), Helicoverpa zea (cornearworm), and Agrotis ipsilon (Black cutworm) nucleic acid and aminoacid sequences. Particularly, the compositions of the inventioncomprise: an M. sexta full-length cDNA herein designated Mag1 (SEQ IDNO:1) and corresponding amino acid sequence (SEQ ID NO:2); an M. sextafull-length cDNA herein designated Rhizoc2 or iimlc.pk003.f3 (SEQ IDNO:3) and corresponding amino acid sequence (SEQ ID NO:4); an M. sextapartial cDNA herein designated iiglc.pk004.f3 (SEQ ID NO:5) andcorresponding amino acid sequence (SEQ ID NO:6); an M. sexta partialcDNA herein designated imilc.pk001.h7 (SEQ ID NO:7) and correspondingamino acid sequence (SEQ ID NO:8); an M. sexta partial cDNA hereindesignated imilc.pk002.m21 (SEQ ID NO:9) and corresponding amino acidsequence (SEQ ID NO:10); an M. sexta full-length cDNA herein designatedRhizoc1 (SEQ ID NO:11) and corresponding amino acid sequence (SEQ IDNO:12); an M. sexta full-length cDNA herein designated Fus1 (SEQ IDNO:13) and corresponding amino acid sequence (SEQ ID NO:14); and an M.sexta full-length cDNA herein designated Rhizoc3 (SEQ ID NO:15) andcorresponding amino acid sequence (SEQ ID NO:16).

The mature Mag1 polypeptide was isolated from the hemolymph of M. sextalarvae induced by injection of the plant pathogenic fungus M. grisea.The Mag1 precursor polypeptide consists of 206 amino acids. Thispolypeptide belongs to a broad class of insect immune proteins known asattacins that were originally isolated from Hyalophora cecropia. A Mag1precursor polypeptide-encoding cDNA (SEQ ID NO:1) was subsequentlyisolated from a cDNA library derived from the fatbodies of pathogeninduced M. sexta. The Mag1 precursor polypeptide shares 78% sequencesimilarity with attacin E/F precursor (SEQ ID NO:19, FIG. 1).

Attacin proteins are induced upon injection of insects (mostlylepidopteran species) with bacteria, and have been demonstrated topossess antibacterial properties (Kockum et al. (1984) EMBO J.3:2071-2075; Engstrom et al. (1984) EMBO J. 3:2065-2070; Engstrom et al.(1984) EMBO J. 3:3347-3351; Bowman et al. (1985) Dev. Comp. Immunol.9:551-558; Sun et al. (1991) Eur. J. Biochem. 196:247-254. The Mag1polypeptide was induced by injection of an insect with a plantpathogenic fungus rather than by induction with a bacteria. Furthermore,the isolated Mag1 polypeptide demonstrates fungicidal activity at lowconcentrations against the plant pathogen M. grisea (see Example 1).

In addition, the polypeptides set forth in SEQ ID NOS:6, 8, and 10, andencoded by the cDNA clones, iiglc.pk004.f3,imilc.pk001.h7, andimilc.pk002.m21, respectively, are also attacin homologs. Thesepolypeptides display about 48 to 62.3% sequence identity to the Mag1polypeptide (SEQ ID NO:2) (see FIG. 2). These cDNA clones were isolatedfrom M. grisea (iiglc.pk004.f3) and B. bassiana (imilc.pk001.h7 andimilc.pk002.m21) induced M. sexta derived cDNA libraries.

Similar to the Mag1 precursor polypeptide, the Rhizoc2 (SEQ ID NO:3)precursor polypeptide also shares sequence homology to the attacin classof proteins. The Rhizoc2 precursor polypeptide shares 75% sequencesimilarity and 68% sequence identity with the attacin E/F precursorprotein shown in FIG. 1 (SEQ ID NO:19). The cDNA encoding the Rhizoc2precursor polypeptide (SEQ ID NO:3) was isolated from a cDNA libraryderived from the fatbodies of R. solani induced M. sexta. The Rhizoc2precursor polypeptide consists of 196 amino acids and the maturepolypeptide demonstrates fungicidal activity at low concentrationsagainst the plant pathogen R. solani (see Example 1). The partial cDNAimilc.pk001.h7 identified from a B. bassiana induced M. sexta library isa fragment of the Rhizoc2 sequence.

Another polypeptide, designated Rhizoc1, with homology to the lebocinclass of insect immune proteins, was similarly isolated from thehemolymph of M. sexta larvae induced by injection of the plantpathogenic fungus R. solani. A Rhizoc1 precursor polypeptide-encodingcDNA (SEQ ID NO:11) was subsequently isolated from a cDNA libraryderived from the fatbodies of M. grisea induced M. sexta. The Rhizoc1precursor polypeptide consists of 142 amino acids and shares 65%sequence similarity and 61% sequence identity with lebocin 4 precursorprotein (Accession No: JC5666).

The Rhizoc1 polypeptide demonstrates fungicidal activity at lowconcentrations against the plant pathogens R. solani and F.verticilloides (see Example 1). Unlike other members of the lebocinclass of polypeptides, the Rhizoc1 polypeptide was induced uponinjection of an insect with a plant fungal pathogen, rather than byinduction with a bacteria. Indeed, other lebocin polypeptides have beendemonstrated to possess antibacterial rather than fungicidal properties(Hara and Yamakawa (1995) Biochem. J 310:651-656; Chowdhury, S. et al.(1995) Biochem. Biophys. Res. Com. 214:271-278; and Furukawa, S. et al.(1997) Biochem. Biophys. Res. Com. 238:769-774).

Additional Rhizoc1 homologs have been identified. The nucleotidesequences of the Rhizoc1 homologs are set forth in SEQ ID NOS:27, 33,45, 48, 51, 72, 81, and 84. The amino acid sequences of the Rhizoc1homologs are set forth in SEQ ID NOS:28, 29, 34, 35, 46, 47, 49, 50, 52,53, 73, 74, 82, 83, 85, and 86.

A mature polypeptide designated Fus1 was isolated from the hemolymph ofM. sexta larvae induced by injection of the plant pathogenic fungus F.verticilloides. This polypeptide demonstrates fungicidal activity at lowconcentrations against the plant pathogen F. verticilloides (see Example1). A cDNA encoding the mature Fus1 polypeptide and part of the signalsequence (SEQ ID NO:13) was subsequently isolated from a cDNA libraryderived from the fatbodies of M. grisea induced M. sexta.

The Fus1 polypeptide of the invention is homologous to several proteinsisolated from insect species that belong to the class of proteins knownas the serine protease inhibitors (Frobius et al. (2000) Eur. J.Biochem. 267:2046-2053; Ramesh et al. (1988) J. Biol. Chem.263:11523-1127; and Sasaki, T (1988) Biol. Chem. 369:1235-1241). TheFus1 polypeptide has about 47% sequence similarity to these proteins.The polypeptides identified by Frobius et al. were isolated fromGalleria mellonella hemolymph after injection of larvae with a yeastpolysaccharide preparation and demonstrate inhibition of serineproteases from the entomopathogenic fungus, Metarhizium anisopliae, aninsect pathogen. A codon-biased Fus1 nucleotide sequence linked to theBAA signal sequence has been created. The codon-biased Fus1 nucleotidesequence was developed according to the codon bias of M. sexta. Thecodon-biased BAA-Fus1 nucleotide sequence is set forth in SEQ ID NO:120and the codon-biased Fus1 sequence is set forth in SEQ ID NO:122. Theamino acid sequence of the BAA-Fus1 polypeptide is set forth in SEQ IDNO:121 and SEQ ID NO:123.

Additional Fus1 homologs have been identified. The nucleotide sequencesof the Fus1 homologs are set forth in SEQ ID NOS:21, 36, and 78. Theamino acid sequences of the Fus1 homologs are set forth in SEQ IDNOS:22, 23, 37, 38, 79, and 80.

A mature polypeptide designated, Rhizoc3, was isolated from thehemolymph of M. sexta larvae induced by injection of the plantpathogenic fungus R. solani. This polypeptide demonstrates fungicidalactivity at low concentrations against the plant pathogen R. solani (seeExample 1).

A Rhizoc3 precursor polypeptide encoding cDNA (SEQ ID NO:15) wassubsequently isolated from a cDNA library derived from the fatbodies ofM. grisea induced M. sexta. The Rhizoc3 precursor polypeptide consistsof 61 amino acids and does not demonstrate sequence homology to anyknown proteins.

Homologs of Fus4 have been identified. The nucleotide sequences of theFus4 homologs are set forth in SEQ ID NOS:24, 30, 39, 42, 54, 57, 60,63, 66, 69, 75, 87, 90, and 93. The amino acid sequences of the Fus4homologs are set forth in SEQ ID NOS:25, 26, 31, 32, 40, 41, 43, 44, 55,56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 76, 77, 88, 89, 91, 92, 94,and 95.

Additional polypeptides active against Fusarium species have beenidentified from Agrotis ipsilon. The Fus6, Fus7, Fus8, Fus9, and Fus10nucleotide sequences are set forth in SEQ ID NOS:100, 102, 104, 106,108, 110, 112, 114, 116, and 118. The amino acid sequences of the Fus6,Fus7, Fus8, Fus9, and Fus10 polypeptides are set forth in SEQ IDNOS:101, 103, 105, 107, 109, 111, 113, 115, 117, and 119.

A codon-biased Fus2 nucleotide sequence linked to the BAA signalsequence has been created. The codon-biased BAA-Fus2 nucleotide sequenceis set forth in SEQ ID NO:124 and the codon-biased Fus2 sequence is setforth in SEQ ID NO:126. The amino acid sequence of the BAA-Fus2polypeptide is set forth in SEQ ID NO:125 and SEQ ID NO:127.

The polypeptides encoded by the nucleotide sequences of the inventionmay be processed into mature peptides as discussed elsewhere herein. Theregion from nucleotide 169 to nucleotide 298 of SEQ ID NO:11 encodes themature Rhizoc1 peptide. The region from nucleotide 58 to nucleotide 624of SEQ ID NO:3 encodes the mature Rhizoc2 peptide. The region fromnucleotide 86 to nucleotide 208 of SEQ ID NO:15 encodes the matureRhizoc3 peptide. The region from nucleotide 46 to nucleotide 216 of SEQID NO:13 encodes the mature Fus1 peptide. The nucleotide sequence setforth in SEQ ID NO:102 encodes the mature Fus6 peptide, the amino acidsequence of which, is set forth in SEQ ID NO:103. The nucleotidesequence set forth in SEQ ID NO:106 encodes the mature Fus7 peptide, theamino acid sequence of which, is set forth in SEQ ID NO:107. Thenucleotide sequence set forth in SEQ ID NO:110 encodes the mature Fus8peptide, the amino acid sequence of which, is set forth in SEQ IDNO:111. The nucleotide sequence set forth in SEQ ID NO:114 encodes themature Fus9 peptide, the amino acid sequence of which, is set forth inSEQ ID NO:115. The nucleotide sequence set forth in SEQ ID NO:118encodes the mature Fus 10 peptide, the amino acid sequence of which, isset forth in SEQ ID NO:119.

Fragments and variants of the disclosed nucleotide sequences andpolypeptides encoded thereby are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence or a portion of the amino acid sequence. Fragments of anucleotide sequence may encode polypeptide fragments that retain thebiological activity of the native protein and hence possessantimicrobial and/or fungicidal activity. By “antimicrobial activity” or“fungicidal activity” is intended the ability to suppress, control,and/or kill the invading pathogenic microbe or fungus, respectively. Acomposition of the invention that possesses antimicrobial or fungicidalactivity will reduce the disease symptoms resulting from microbial orfungal pathogen challenge by at least about 5% to about 50%, at leastabout 10% to about 60%, at least about 30% to about 70%, at least about40% to about 80%, or at least about 50% to about 90% or greater.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the invention.

Alternatively, fragments of a nucleotide sequence of the invention mayencode polypeptide fragments that are antigenic, thus, they are capableof eliciting an immune response. An “antigenic polypeptide” is hereindefined as a polypeptide that is capable of generating an antibody.Antigenic polypeptide fragments of the disclosed amino acid sequencesare also encompassed by the invention.

A nucleotide fragment of SEQ ID NO:1 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:2 (Mag1),will encode at least 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150,or 200 contiguous amino acids, or up to the total number of amino acids(206) present in SEQ ID NO:2.

A nucleotide fragment of SEQ ID NO:3 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:4(Rhizoc2), will encode at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190contiguous amino acids, or up to the total number of amino acids (196)present in SEQ ID NO:4.

A nucleotide fragment of SEQ ID NO:5 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:6(iiglc.pk004.f3), will encode at least 25, 30, 35, 40, 45, 50, 55, 60,or 70 contiguous amino acids, or up to the total number of amino acids(80) present in SEQ ID NO:6.

A nucleotide fragment of SEQ ID NO:7 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:8(imilc.pk001.h7), will encode at least 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, or 110 contiguous amino acids, or up to the totalnumber of amino acids (111) present in SEQ ID NO:8.

A nucleotide fragment of SEQ ID NO:9 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:10(imilc.pk002.m21), will encode at least 25, 30, 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 110, 120, 130, or 140 contiguous amino acids, or up tothe total number of amino acids (148) present in SEQ ID NO:10.

A nucleotide fragment of SEQ ID NO:11 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:12(Rhizoc1), will encode at least 15, 20, 25, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 110, 120, 130, or 140 contiguous amino acids, or up to thetotal number of amino acids (142) present in SEQ ID NO:12.

A nucleotide fragment of SEQ ID NO:13 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:14 (Fus1),will encode at least 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70contiguous amino acids, or up to the total number of amino acids (71)present in SEQ ID NO:14.

A nucleotide fragment of SEQ ID NO:15 that encodes a biologically activeor antigenic portion of the amino acid sequence of SEQ ID NO:16(Rhizoc3), will encode at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, or 60 contiguous amino acids, or up to the total number of aminoacids (61) present in SEQ ID NO:16.

A nucleotide fragment of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 21, 24,27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78,81, 84, 87, 90, 93, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, or 126 that encodes a biologically active or antigenicportion of the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46,47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73,74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 101, 103,105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, or 127, willencode at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 contiguousamino acids, or up to the total number of amino acids present in SEQ IDNO:2, 4, 6, 8, 10, 12, 14, 16, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35,37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62,64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89,91, 92, 94, 95, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121,123, 125, or 127.

A biologically active or antigenic portion of a polypeptide sequence setforth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 22, 23, 25, 26, 28, 29,31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56,58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83,85, 86, 88, 89, 91, 92, 94, 95, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, or 127 can be prepared by isolating a portionof one of the nucleotide sequences set forth in SEQ ID NO:1, 3, 5, 7, 9,11, 13, 15, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63,66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, or 126, expressing the encodedportion of the polypeptide (e.g., by recombinant expression in vitro),and assessing the activity of the encoded portion of the polypeptide.

Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode fragment polypeptidesretaining biological activity. Thus, fragments of a nucleotide sequencemay range from at least about 15 nucleotides, about 30 nucleotides,about 50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence encoding the polypeptides of the invention.

Fragments of the nucleotide sequence set forth in SEQ ID NO:1, fromnucleotide 4 to 621, may range from at least 15, 20, 30, 40, 50, 60, 70,80, 90, 100, 125, 150, 200, 300, 400, or 500 contiguous nucleotides, orup to the total number of nucleotides (618) present in SEQ ID NO:1 thatencode SEQ ID NO:2 (Mag1).

Fragments of the nucleotide sequence set forth in SEQ ID NO:3, fromnucleotide 34 to 624, may range from at least 15, 20, 30, 40, 50, 60,70, 80, 90, 100, 125, 150, 200, 300, 400, or 500 contiguous nucleotides,or up to the total number of nucleotides (588) present in SEQ ID NO:3that encode SEQ ID NO:4 (Rhizoc2).

Fragments of the nucleotide sequence set forth in SEQ ID NO:5(iiglc.pk004.f3), from nucleotide 4 to 249, may range from at least 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, or 200 contiguousnucleotides, or up to the total number of nucleotides (240) present inSEQ ID NO:5 that encode SEQ ID NO:6.

Fragments of the nucleotide sequence set forth in SEQ ID NO:7(imilc.pk001.h7), from nucleotide 4 to 336, may range from at least 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, or 300contiguous nucleotides, or up to the total number of nucleotides (333)present in SEQ ID NO:7 that encode SEQ ID NO:8. SEQ ID NO:7 is afragment of the nucleotide sequence set forth in SEQ ID NO:3.

Fragments of the nucleotide sequence set forth in SEQ ID NO:9(imilc.pk002.m21), from nucleotide 4 to 447, may range from at least 15,20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, or400 contiguous nucleotides, or up to the total number of nucleotides(444) present in SEQ ID NO:9 that encode SEQ ID NO:10.

Fragments of the nucleotide sequence set forth in SEQ ID NO:11(Rhizoc1), from nucleotide 28 to 456, may range from at least 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 250, 300, 350, or 400contiguous nucleotides, or up to the total number of nucleotides (426)present in SEQ ID NO:11 that encode SEQ ID NO:12.

Fragments of the nucleotide sequence set forth in SEQ ID NO:13 (Fus1),from nucleotide 22 to 237, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, or 200 contiguous nucleotides, or upto the total number of nucleotides (216) present in SEQ ID NO:13 thatencode SEQ ID NO:14.

Fragments of the nucleotide sequence set forth in SEQ ID NO:15(Rhizoc3), from nucleotide 23 to 208, may range from at least 15, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, or 150 contiguous nucleotides, orup to the total number of nucleotides (183) present in SEQ ID NO:15 thatencode SEQ ID NO:16.

Fragments of the nucleotide sequence set forth in SEQ ID NO:21, 24, 27,30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81,84, 87, 90, or 93 may range from at least 15, 20, 30, 40, 50, 60, 70,80, 90, 100, 125, or 150 contiguous nucleotides, or up to the totalnumber of nucleotides present in SEQ ID NO:21, 24, 27, 30, 33, 36, 39,42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, or93 that encode SEQ ID NO:22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38,40, 41, 43, 44, 46, 47, 49, 50, 52, 53, 55, 56, 58, 59, 61, 62, 64, 65,67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92,94, or 95, respectively.

Fragments of the nucleotide sequence set forth in SEQ ID NO:100 (Fus6),from nucleotide 1 to 195, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 180, 185, 190, or 195 contiguousnucleotides, or up to the total number of nucleotides (358) present inSEQ ID NO:100 that encode SEQ ID NO:101.

Fragments of the nucleotide sequence set forth in SEQ ID NO:104 (Fus7),from nucleotide 1 to 195, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 180, 185, 190, or 195 contiguousnucleotides, or up to the total number of nucleotides (387) present inSEQ ID NO:104 that encode SEQ ID NO:105.

Fragments of the nucleotide sequence set forth in SEQ ID NO:108 (Fus8),from nucleotide 1 to 195, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 180, 185, 190, or 195 contiguousnucleotides, or up to the total number of nucleotides (361) present inSEQ ID NO:108 that encode SEQ ID NO:109.

Fragments of the nucleotide sequence set forth in SEQ ID NO:112 (Fus9),from nucleotide 1 to 195, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 180, 185, 190, 195, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, or 291 contiguous nucleotides, or upto the total number of nucleotides (466) present in SEQ ID NO:112 thatencode SEQ ID NO:113.

Fragments of the nucleotide sequence set forth in SEQ ID NO:116 (Fus10),from nucleotide 1 to 195, may range from at least 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 180, 185, 190, 195, 200, 210, or 220contiguous nucleotides, or up to the total number of nucleotides (372)present in SEQ ID NO:116 that encode SEQ ID NO:117.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

A protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, (bydry weight) of contaminating protein. When the protein of the inventionor biologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the polypeptides of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but that still encode a polypeptide of theinvention. Generally, variants of a particular nucleotide sequence ofthe invention will have at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequenceidentity to that particular nucleotide sequence as determined bysequence alignment programs described elsewhere herein using defaultparameters.

By “variant” polypeptide is intended a polypeptide derived from thenative polypeptide by deletion (so-called truncation) or addition of oneor more amino acids to the N-terminal and/or C-terminal end of thenative polypeptide; deletion or addition of one or more amino acids atone or more sites in the native polypeptide; or substitution of one ormore amino acids at one or more sites in the native polypeptide. Variantpolypeptides encompassed by the present invention are biologicallyactive, that is, they continue to possess the desired biologicalactivity of the native polypeptide, hence they will continue to possessantimicrobial and/or fungicidal activity. Such variants may result from,for example, genetic polymorphism or from human manipulation.

Biologically active variants of a native polypeptide of the inventionwill have at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the aminoacid sequence for the native polypeptide as determined by sequencealignment programs described elsewhere herein using default parameters.A biologically active variant of a polypeptide of the invention maydiffer from that polypeptide by as few as 1-15 amino acid residues, asfew as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1amino acid residue.

Biological activity of the polypeptides of the present invention can beassayed by any method known in the art (see for example, U.S. Pat. No.5,614,395; Thomma et al. (1998) Plant Biology 95:15107-15111; Liu et al.(1994) Plant Biology 91:1888-1892; Hu et al. (1997) Plant Mol. Biol.34:949-959; Cammue et al. (1992) J. Biol Chem. 267:2228-2233; andThevissen et al. (1996) J. Biol Chem. 271:15018-15025, all of which areherein incorporated by reference).

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the polypeptides ofthe invention can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, thepolypeptides of the invention encompass both naturally occurringpolypeptides as well as variations and modified forms thereof. Suchvariants will continue to possess the desired antimicrobial, or in somecases, fungicidal activity. Obviously, the mutations that will be madein the DNA encoding the variant must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure. See, EP Patent ApplicationPublication No. 75,444.

The deletions, insertions, and substitutions of the polypeptidesequences encompassed herein are not expected to produce radical changesin the characteristics of the polypeptide. However, when it is difficultto predict the exact effect of the substitution, deletion, or insertionin advance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays for antimicrobialand/or fungicidal activity as referenced supra.

Variant nucleotide sequences and polypeptides also encompass sequencesand polypeptides derived from a mutagenic and recombinogenic proceduresuch as DNA shuffling. With such a procedure, one or more differentcoding sequences in the nucleic acid molecules described in SEQ ID NO:1,3, 5, 7, 9, 11, 13, 15, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54,57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, or 126 can be manipulatedto create a new polypeptides possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled betweenthe nucleic acid molecules of the invention and other knownantimicrobial encoding nucleotide sequences to obtain a new nucleotidesequence coding for a polypeptide with an improved property of interest,such as increased antimicrobial and/or fungicidal properties at lowerpolypeptide concentrations or specificity for particular plant pathogensas well as, for example, specificity for a particular plant fungalpathogen including, but not limited to, pathogens such as M. grisea andF. verticilloides. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (17994) Nature 370:389-391; Crameri et al.(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly otherinsects. In this manner, methods such as PCR, hybridization, and thelike can be used to identify such sequences based on their sequencehomology to the sequences set forth herein. Sequences isolated based ontheir sequence identity to the full-length nucleotide sequences setforth herein or to fragments thereof are encompassed by the presentinvention. Such sequences include sequences that are orthologs of thedisclosed sequences. By “orthologs” is intended genes derived from acommon ancestral gene and which are found in different species as aresult of speciation. Genes found in different species are consideredorthologs when their nucleotide sequences and/or their encodedpolypeptide sequences share substantial identity as defined elsewhereherein. Functions of orthologs are often highly conserved among species.Thus, isolated sequences that encode an antimicrobial protein and whichhybridize under stringent conditions to the nucleotide sequencesdisclosed herein, or to fragments thereof, are encompassed by thepresent invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any insect of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the disease resistantsequences of the invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, an entire nucleotide sequence disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to the corresponding nucleotide sequences and messengerRNAs. To achieve specific hybridization under a variety of conditions,such probes include sequences that are unique among the nucleotidesequences of the invention and are preferably at least about 10nucleotides in length, and most preferably at least about 20 nucleotidesin length. Such probes may be used to amplify corresponding sequencesfrom a chosen organism by PCR. This technique may be used to isolateadditional coding sequences from a desired organism or as a diagnosticassay to determine the presence of coding sequences in an organism.Hybridization techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see, for example, Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Thus, isolated sequences that encode for an anti-microbial polypeptideand which hybridize under stringent conditions to a sequence disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridizationis generally less than about 24 hours, usually about 4 to about 12hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,N.Y.). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual(2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS4:11-17; the local homology algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-similarity-method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0); the ALIGN PLUS program (version 3.0,copyright 1997); and GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Version 8 (available from GeneticsComputer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignmentsusing these programs can be performed using the default parameters. TheCLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. TheALIGN and the ALIGN PLUS programs are based on the algorithm of Myersand Miller (1988) supra. A PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4 can be used with the ALIGN programwhen comparing amino acid sequences.

The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin and Altschul (1990) supra. The BLASTfamily of programs that can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTP for peptide query sequences against a peptidedatabase; BLASTX for nucleotide query sequences against protein databasesequences; TBLASTN for protein query sequences against nucleotidedatabase sequences; and TBLASTX for nucleotide query sequences againstnucleotide databases with the translation of all nucleotide sequences toprotein. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a polypeptide of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See www.ncbi.hlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity using GAP Weight of 50 and LengthWeight of 3;% similarity using Gap Weight of 12 and Length Weight of 4,or any equivalent program. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

For purposes of the present invention, comparison of nucleotide orpolypeptide sequences for determination of percent sequence identity tothe nucleotide or polypeptide sequences disclosed herein is preferablymade using the ClustalW program (Version 1.7 or later) with its defaultparameters or any equivalent program. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide or aminoacid residue matches and an identical percent sequence identity whencompared to the corresponding alignment generated by the preferredprogram.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 80%,90%, 95%, or more sequence identity compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity ofpolypeptides encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 60%, morepreferably at least 70%, 80%, 90%, or 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 80%,85%, 90%, or 95% sequence identity to the reference sequence over aspecified comparison window. Preferably, optimal alignment is conductedusing the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol. Biol. 48:443-453. An indication that two peptide sequences aresubstantially identical is that one peptide is immunologically reactivewith antibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

The nucleic acid sequences of the present invention can be expressed ina host cell such as bacteria, fungi, yeast, insect, mammalian, or plantcells. It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of a nucleicacid encoding a polypeptide of the present invention. No attempt todescribe in detail the various methods known for the expression ofpolypeptides in prokaryotes or eukaryotes will be made.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species, or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which comprises a heterologous nucleicacid sequence of the invention. Host cells may be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells. Preferably, host cells are monocotyledonous ordicotyledonous plant cells, particularly rice and maize plant cells.

The disease resistance-conferring sequences of the invention areprovided in expression cassettes or DNA constructs for expression in theplant of interest. The cassette will include 5′ and 3′ regulatorysequences operably linked to a nucleotide sequence of the invention. By“operably linked” is intended a functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the nucleotide sequence corresponding to thesecond sequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame. Thecassette may additionally contain at least one additional gene to becotransformed into the organism. Alternatively, the additional gene(s)can be provided on multiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the disease resistant sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, asignal peptide sequence, a disease resistant DNA sequence of theinvention, and a transcriptional and translational termination regionfunctional in plants. The transcriptional initiation region, thepromoter, may be native or analogous or foreign or heterologous to theplant host. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. By “foreign” is intended that thetranscriptional initiation region is not found in the native plant intowhich the transcriptional initiation region is introduced. As usedherein, a chimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

While it may be preferable to express the sequences using heterologouspromoters, the native promoter sequences may be used. Such constructswould vary expression levels of the disease resistant RNA/protein in theplant or plant cell. Thus, the phenotype of the plant or plant cell isaltered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,or may be derived from another source. Convenient termination regionsare available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See alsoGuerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the nucleotide sequences may be optimized forincreased expression in the transformed host. That is, the nucleotidesequences can be synthesized using plant-preferred codons for improvedexpression in plants. Methods are available in the art for synthesizingplant-preferred nucleotide sequences or genes. See, for example, U.S.Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) NucleicAcids Res. 17:477-498, herein incorporated by reference. Nucleotidesequences have been created that encode Fus1 and Fus2 operably linked toBAA and codon biased for expression in host cells. The BAA-Fus1nucleotide sequence was codon-biased according to M. sexta codon usage.The BAA-Fus2 nucleotide sequence was codon-biased according toStreptomyces coelicolor codon usage. S. coelicolor codon usage patternsresemble the codon usage patterns of many plants. The development of thecodon-biased sequences is described elsewhere herein.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

In certain embodiments of the invention, it is desirable to utilize themature peptide or the nucleotide sequence encoding the mature peptide.Within the cell, proteolytic modifications of amino acid sequences occurfrequently. The proteolytic event removes amino acids from the precursorpolypeptide to yield a mature peptide. The proteolytic processing can behighly sequence specific. Often the precursor peptides are inactivewhile the mature peptides possess the desired activity. Thus, isolationof a peptide based on its activity results in isolation of the active,mature peptide. Discovery of the existence of pre-sequences occurs whenthe nucleotide sequence encoding the mature peptide is identified. Theopen reading frame that encodes the mature peptide also encodes thepresequences that were removed by the cell. Proteolytic maturation ofamino acid sequences occurs in multiple cellular locations including,but not limited to, the endoplasmic reticulum, the cytoplasm, themitochondria, the chloroplasts, the nucleus, the Golgi Apparatus, andthe extracellular matrix. Proteolytic processing of peptides isdiscussed in Creighton, T. E. (1993) Proteins: Structures & MolecularProperties. W. H. Freeman & Co., U.S.A and Alberts et al eds. (1994)Molecular Biology of the Cell. Garland Publishing, Inc., N.Y., hereinincorporated by reference. Rather than rely on a host cell to properlyprocess the polypeptide of the invention, employment of a nucleotidesequence encoding the mature peptide may be desirable.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virusleader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also,Della-Cioppa et al. (1987) Plant Physiol. 84:965-968. Other methodsknown to enhance translation can also be utilized, for example, introns,and the like.

Signal peptides may be fused to the disease resistant nucleotidesequence of the invention to direct transport of the expressed geneproduct out of the cell to the desired site of action in theintercellular space. Examples of signal peptides include those nativelylinked to the Barley alpha amylase protein (BAA), sporamin,oryzacystatin-I, and those from the plant pathogenesis-related proteins,e.g., PR-1, PR-2, etc.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D), and sulfonylureas (SUs). Seegenerally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. NatLAcad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 ( Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred,inducible or other promoters for expression in plants. Such constitutivepromoters include, for example, the core promoter of the Rsyn7 promoterand other constitutive promoters disclosed in WO 99/43838 and U.S. Pat.No. 6,072,050; Scp1 promoter (U.S. Pat. No. 6,072,050), rice actin(McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen etal. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992)Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), Maize h2B (PCT Application SerialNo. WO 99/43797) and the like. Other constitutive promoters include, forexample, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Generally, it will be beneficial to express the gene from an induciblepromoter, particularly from a pathogen-inducible promoter. Suchpromoters include those from pathogenesis-related proteins (PRproteins), which are induced following infection by a pathogen; e.g., PRproteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, forexample, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Ukneset al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol.Virol. 4:111-116. See also WO 99/43819, herein incorporated byreference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. NatL. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhancedantimicrobial polypeptide expression within a particular plant tissue.See, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al.(1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535;Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. Thus, any method, which provides for effectivetransformation/transfection may be employed. Transformation protocols aswell as protocols for introducing nucleotide sequences into plants mayvary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of introducingnucleotide sequences into plant cells and subsequent insertion into theplant genome include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation(Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No.5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No.5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lecl transformation (WO 00/28058). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic has been achieved.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, rice (Oryza sativa),corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and muskmelon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Preferably, plants of the present invention are crop plants (forexample, rice, corn, alfalfa, sunflower, Brassica, soybean, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.).

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding sequences, include such commonly used promoters as thebeta lactamase (penicillinase) and lactose (lac) promoter systems (Changet al. (1977) Nature 198:1056), the tryptophan (trp) promoter system(Goeddel et al. (1980) Nucleic Acids Res. 8:4057) and the lambda derivedP L promoter and N-gene ribosome binding site (Shimatake et al. (1981)Nature 292:128). The inclusion of selection markers in DNA vectorstransfected in E coli. is also useful. Examples of such markers includegenes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a polypeptide of the present inventionare available using Bacillus sp. and Salmonella (Palva et al. (1983)Gene 22:229-235); Mosbach et al. (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a polynucleotide of the presentinvention can be expressed in these eukaryotic systems. In someembodiments, transformed/transfected plant cells, as discussed infra,are employed as expression systems for production of the polypeptides ofthe instant invention.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect, or plant origin. Illustrative cell culturesuseful for the production of the peptides are mammalian cells. A numberof suitable host cell lines capable of expressing intact proteins havebeen developed in the art, and include the HEK293, BHK21, and CHO celllines. Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter (e.g. the CMVpromoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter),an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T Ag poly A additionsite), and transcriptional terminator sequences. Other animal cellsuseful for production of polypeptides of the present invention areavailable, for instance, from the American Type Culture Collection.

Appropriate vectors for expressing polypeptides of the present inventionin insect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See, Schneider(1987) J. Embryol. Exp. Morphol. 27:353-365).

As with yeast, when higher animal or plant host cells are employed,polyadenylation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al.(1983)J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo (1985)DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press,Arlington, Va., pp. 213-238).

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art (Kuchler(1997) Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc.).

Synthesis of heterologous nucleotide sequences in yeast is well known(Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory). Two widely utilized yeasts for production of eukaryoticproteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors,strains, and protocols for expression in Saccharomyces and Pichia areknown in the art and available from commercial suppliers (e.g.,Invitrogen). Suitable vectors usually have expression control sequences,such as promoters, including 3-phosphoglycerate kinase or alcoholoxidase, and an origin of replication, termination sequences and thelike as desired.

A polypeptide of the present invention, once expressed, can be isolatedfrom yeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques, UV absorptionspectra, radioimmunoassay, or other standard immunoassay techniques.

The invention is drawn to a general method for identifying and makingantimicrobial compositions, particularly antifungal compositions. Themethods involve injection of an insect with a suspension of a plantpathogenic fungus to induce insect polypeptides possessing antimicrobialactivity. Such polypeptides are isolated from the insect hemolymph usinga combination of high-resolution liquid chromatography and massspectrophotometry.

The general strategy for the discovery of these insect-derivedantimicrobial peptides involves challenging insects with a selectedplant pathogen and collecting hemolymph and fat body samples at varioustimes post-induction. For example, hemolymph and fat body samples can becollected at about 8 hour, 16 hour, 24 hour, or 48 hour intervals. It isrecognized that any method for protein separation and identification maybe used to isolate peptides and the corresponding nucleic acidsequences.

While not bound by any particular method, identification ofantimicrobial peptides active against the target pathogen may beachieved using an integrated proteomic, genomic, and miniaturizedbioassay approach. This approach consists of separation of hemolymphisolated from induced insects. Any method of separation can be usedincluding HPLC separation. Fractions from HPLC-aided separation may beseparated into 30-second fractions in a microtiter plate format, i.e.,96 well microtiter plate. Fractions collected in this manner are drieddown and directly used in a fungal growth assay (FGA) in which the driedfractions are resuspended in 100 μl of half strength potato dextrosebroth containing a suspension of the target fungal pathogen. Fractionsthat contain antimicrobial peptides are identified in the FGA by theirability to inhibit fungal growth after several hours, generally 24 to 48hours. These fractions are subjected to further purification in order toisolate individual peptides and the specific peptide responsible for theobserved activity is determined by FGA. This peptide is subsequentlyN-terminally sequenced and its molecular weight determined by massspectrometry to provide information to identify the corresponding genefrom sequence data derived from the corresponding insect cDNA libraries.The complete amino acid sequence of the peptide is determined bytranslation of the nucleic acid sequence and the mature peptideidentified based on both N-terminal sequence and molecular weightinformation.

The defensive agents of the invention encompasses the mature activepeptides as well as unprocessed or prepro-forms of the peptides. Where amature peptide has been isolated, the prepro sequence, or signalsequence, can be obtained by a number of general molecular biologytechniques known in the art.

As indicated, the defensive agents may be isolated from any insect ofinterest. Of particular interest are insects living in harshenvironments and insects that are natural plant predators. While anyinsect may be utilized, it may be beneficial to use insect predators ofa particular plant of interest. For example, to obtain defensive agentsfor use in maize, while any insect may be used, maize insect predatorsmay be beneficial.

Although a defensive agent may be induced by a particular pathogen, itis anticipated that the defensive agent may be effective against one ormore additional pathogens, including but not limited to, any of thepathogens listed above.

The polypeptides are tested for antimicrobial activity using in vitroassays as described elsewhere herein. Isolated antimicrobialpolypeptides are subjected to proteolysis, and the amino termini of theresulting proteolytic fragments are sequenced. Degenerateoligonucleotides encoding the amino terminal sequence tags are used toidentify the antimicrobial polypeptide-encoding cDNA's fromcorresponding pathogen induced insect cDNA libraries. The nucleic acidmolecules encoding the antimicrobial polypeptides are used for thetransformation of plant cells to generate plants with enhanced diseaseresistance. Additionally, the compositions of the invention can be usedto generate formulations possessing disease resistance activities.

Methods for increasing pathogen resistance in a plant are provided. Themethods involve stably transforming a plant with a DNA constructcomprising a nucleotide sequence of a defensive agent of the inventionoperably linked to promoter that drives expression in a plant. Suchmethods may find use in agriculture particularly in limiting the impactof plant fungal pathogens on crop plants. The antimicrobial nucleotidesequences comprise the insect nucleic acid molecules of the inventionand functional variants and fragments thereof. The choice of promoterwill depend on the desired timing and location of expression of theantimicrobial nucleotide sequences. Promoters of the invention includeconstitutive, inducible, and tissue-preferred promoters.

As discussed above, the nucleotide sequences of the invention encodepolypeptides with antimicrobial properties, particularly fungicidalproperties. Hence, the sequences of the invention may enhance transgenicplant disease resistance by disrupting cellular function of plantpathogens, particularly plant fungal pathogens. However, it isrecognized that the present invention is not dependent upon a particularmechanism of defense. Rather, the compositions and methods of theinvention work to increase resistance of the plant to pathogensindependent of how that resistance is increased or achieved.

The methods of the invention can be used with other methods available inthe art for enhancing disease resistance in plants. Similarly, theantimicrobial compositions described herein may be used alone or incombination with other nucleotide sequences, polypeptides, or agents toprotect against plant diseases and pathogens. Although any one of avariety of second nucleotide sequences may be utilized, specificembodiments of the invention encompass those second nucleotide sequencesthat, when expressed in a plant, help to increase the resistance of aplant to pathogens.

Proteins, peptides, and lysozymes that naturally occur in insects(Jaynes et al. (1987) Bioassays 6:263-270), plants (Broekaert et al.(1997) Critical Reviews in Plant Sciences 16:297-323), animals (Vunnamet al. (1997) J. Peptide Res. 49:59-66), and humans (Mitra and Zang(1994) Plant Physiol. 106:977-981; Nakajima et al. (1997) Plant CellReports 16:674-679) are also a potential source of plant diseaseresistance. Examples of such plant resistance-conferring sequencesinclude those encoding sunflower rhoGTPase-Activating Protein (rhoGAP),lipoxygenase (LOX), Alcohol Dehydrogenase (ADH), andSclerotinia-Inducible Protein-1 (SCIP-1) described in U.S. applicationSer. No. 09/714,767, herein incorporated by reference. These nucleotidesequences enhance plant disease resistance through the modulation ofdevelopment, developmental pathways, and the plant pathogen defensesystem. Other plant defense proteins include those described in WO99/43823 and WO 99/43821, all of which are herein incorporated byreference. It is recognized that such second nucleotide sequences may beused in either the sense or antisense orientation depending on thedesired outcome.

In one embodiment of the invention, at least one expression cassettecomprising a nucleic acid molecule encoding the Mag1 polypeptide setforth in SEQ ID NO:2 is stably incorporated into a rice plant host, toconfer on the plant enhanced disease resistance to fungal pathogens,particularly the pathogen M. grisea. While the choice of promoter willdepend on the desired timing and location of expression of the Mag1nucleotide sequence, preferred promoters include constitutive andpathogen-inducible promoters. By “inducible” is intended the ability ofthe promoter sequence to regulate expression of an operably linkednucleotide sequence in response to a stimulus. In the case of apathogen-inducible promoter, regulation of expression will be inresponse to a pathogen-derived stimulus.

Another embodiment of the invention involves the stable incorporation ofat least one expression cassette comprising a nucleotide sequenceencoding at least one of the Rhizoc1 polypeptide set forth in SEQ IDNO:12, the Rhizoc2 polypeptide set forth in SEQ ID NO:4, or the Rhizoc3polypeptide set forth in SEQ ID NO:16 into a rice plant host to conferon the plant enhanced disease resistance to fungal pathogens,particularly the pathogen R. solani. While the choice of promoter willdepend on the desired timing and location of expression of the Mag1nucleotide sequence, preferred promoters include constitutive andpathogen-inducible promoters.

An additional embodiment of the invention involves the stableincorporation of at least one expression cassette comprising anucleotide sequence encoding at least one of the Rhizoc1 polypeptide setforth in SEQ ID NO:12 or the Fus1 polypeptide set forth in SEQ ID NO:14into a corn plant host to confer on the plant enhanced diseaseresistance to fungal pathogens, particularly the pathogen F.verticilloides. In an embodiment the nucleotide sequence is acodon-biased sequence, such as the codon-biased sequence set forth inSEQ ID NO:122, 124, 126, or 128. While the choice of promoter willdepend on the desired timing and location of expression of the Mag1nucleotide sequence, preferred promoters include constitutive andpathogen-inducible promoters.

In an embodiment of the invention, the polypeptides of the invention canbe formulated with an acceptable carrier into an antimicrobialcomposition(s) that is for example, a suspension, a solution, anemulsion, a dusting powder, a dispersible granule, a wettable powder,and an emulsifiable concentrate, an aerosol, an impregnated granule, anadjuvant, or a coatable paste, and also in encapsulations, for example,polymer substances.

In another embodiment, the defensive agents comprise isolatedpolypeptides of the invention. The defensive agents of the inventionfind use in the decontamination of plant pathogens during the processingof grain for animal or human food consumption; during the processing offeedstuffs, and during the processing of plant material for silage. Inthis embodiment, the defensive agents of the invention are presented tograin, plant material for silage, or a contaminated food crop, or duringan appropriate stage of the processing procedure, in amounts effectivefor anti-microbial activity. The compositions can be applied to theenvironment of a plant pathogen by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment, or dustingat the time when the plant pathogen has begun to appear or before theappearance of pests as a protective measure. It is recognized that anymeans to bring the defensive agent polypeptides in contact with theplant pathogen can be used in the practice of the invention.

Methods are provided for controlling plant pathogens comprising applyinga decontaminating amount of a polypeptide or composition of theinvention to the environment of the plant pathogen. The polypeptides ofthe invention can be formulated with an acceptable carrier into acomposition(s) that is, for example, a suspension, a solution, anemulsion, a dusting powder, a dispersible granule, a wettable powder, anemulsifiable concentrate, an aerosol, an impregnated granule, anadjuvant, a coatable paste, and also encapsulations in, for example,polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bacteriocides, nematocides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants, or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular targetmycotoxins. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers. Theactive ingredients of the present invention are normally applied in theform of compositions and can be applied to the crop area or plant to betreated, simultaneously or in succession, with other compounds. In someembodiments, methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention (whichcontains at least one of the proteins of the present invention) arefoliar application, seed coating, and soil application.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate, or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate, oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include, but are not limited to, inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as concentrate of primary composition, whichrequires dilution with a suitable quantity of water or other diluentbefore application. The decontaminating concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly.

In a further embodiment, the compositions, as well as the polypeptidesof the present invention can be treated prior to formulation to prolongthe activity when applied to the environment of a plant pathogen as longas the pretreatment is not deleterious to the activity. Such treatmentcan be by chemical and/or physical means as long as the treatment doesnot deleteriously affect the properties of the composition(s). Examplesof chemical reagents include, but are not limited to, halogenatingagents; aldehydes such as formaldehyde and glutaraldehyde;anti-infectives, such as zephiran chloride; alcohols, such asisopropanol and ethanol; and histological fixatives, such as Bouin'sfixative and Helly's fixative (see, for example, Humason (1967) AnimalTissue Techniques (W. H. Freeman and Co.).

In an embodiment of the invention, the compositions of the inventioncomprise a microbe having stably integrated the nucleotide sequence of adefensive agent. The resulting microbes can be processed and used as amicrobial spray. Any suitable microorganism can be used for thispurpose. See, for example, Gaertner et al. (1993) in Advanced EngineeredPesticides, Kim (Ed.). In one embodiment, the nucleotide sequences ofthe invention are introduced into microorganisms that multiply on plants(epiphytes) to deliver the defensive agents to potential target crops.Epiphytes can be, for example, gram-positive or gram-negative bacteria.

It is further recognized that whole, i.e., unlysed, cells of thetransformed microorganism can be treated with reagents that prolong theactivity of the polypeptide produced in the microorganism when themicroorganism is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside themicroorganism for presentation to the target plant.

In this manner, a gene encoding a defensive agent of the invention maybe introduced via a suitable vector into a microbial host, and saidtransformed host applied to the environment, plants, or animals.Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest may be selected for transformation. Thesemicroorganisms are selected so as to be capable of successfullycompeting in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the detoxifying polypeptide, and for improved protectionof the enzymes of the invention from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, Aureobasidium, andGliocladium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, Clavibacter xyli, and Azotobacter vinlandii; and phytosphereyeast species such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesrosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

Illustrative prokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

In an embodiment of the invention, the defensive agents of the inventionmay be used as a pharmaceutical compound for treatment of fungal andmicrobial pathogens in humans and other animals. Diseases and disorderscaused by fungal and microbial pathogens include but are not limited tofungal meningoencephalitis, superficial fungal infections, ringworm,Athlete's foot, histoplasmosis, candidiasis, thrush, coccidioidoma,pulmonary cryptococcus, trichosporonosis, piedra, tinea nigra, fungalkeratitis, onychomycosis, tinea capitis, chromomycosis, aspergillosis,endobronchial pulmonary aspergillosis, mucormycosis,chromoblastomycosis, dermatophytosis, tinea, fusariosis, pityriasis,mycetoma, pseudallescheriasis, and sporotrichosis.

The compositions of the invention may be used as pharmaceuticalcompounds to provide treatment for diseases and disorders associatedwith, but not limited to, the following fungal pathogens: Histoplasmacapsulatum, Candida spp. (C. albicans, C. tropicalis, C. parapsilosis,C. guilliermondii, C. glabrata/Torulopsis glabrata, C. krusei, C.lusitaniae), Aspergillus fumigatus, A. flavus, A. niger, Rhizopus spp.,Rhizomucor spp., Cunninghamella spp., Apophysomyces spp., Saksenaeespp., Mucor spp., and Absidia spp. Efficacy of the compositions of theinvention as anti-fungal treatments may be determined throughanti-fungal assays known to one of skill in the art.

The defensive agents may be administered to a patient through numerousmeans. Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

“Treatment” is herein defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A “therapeutic agent”includes, but is not limited to, small molecules, peptides, antibodies,ribozymes and antisense oligonucleotides.

The defensive agents of the invention can be used for any applicationincluding coating surfaces to target microbes. In this manner, targetmicrobes include human pathogens or microorganisms. Surfaces that mightbe coated with the defensive agents of the invention include carpets andsterile medical facilities. Polymer bound polypeptides of the inventionmay be used to coat surfaces. Methods for incorporating compositionswith anti-microbial properties into polymers are known in the art. SeeU.S. Pat. No. 5,847,047, herein incorporated by reference.

An isolated polypeptide of the invention can be used as an immunogen togenerate antibodies that bind defensive agents using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthdefensive agents can be used or, alternatively, the invention providesantigenic peptide fragments of defensive agents for use as immunogens.The antigenic peptide of a defensive agent comprises at least 8,preferably 10, 15, 20, or 30 amino acid residues of the amino acidsequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 22, 23, 25, 26,28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, 47, 49, 50, 52, 53,55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80,82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 96, 97, 98, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, or 127, andencompasses an epitope of a defensive agent such that an antibody raisedagainst the peptide forms a specific immune complex with theanti-microbial polypeptides. Preferred epitopes encompassed by theantigenic peptide are regions of defensive agents that are located onthe surface of the protein, e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-defensiveagent polyclonal and monoclonal antibodies that bind a defensive agent.Polyclonal defensive agent-like antibodies can be prepared by immunizinga suitable subject (e.g., rabbit, goat, mouse, or other mammal) with adefensive agent-like immunogen. The anti-defensive agent antibody titerin the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized anti-microbial polypeptides. At an appropriate timeafter immunization, e.g., when the anti-defensive agent antibody titersare highest, antibody-producing cells can be obtained from the subjectand used to prepare monoclonal antibodies by standard techniques, suchas the hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497, the human B cell hybridoma technique (Kozboret al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole etal. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld andSell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Coligan et al., eds. (1994) Current Protocols in Immunology(John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension InBiological Analyses (Plenum Publishing Corp., New York; and Lerner(1981) Yale J. Biol Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-defensive agent-like antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with a defensive agent tothereby isolate immunoglobulin library members that bind the defensiveagent. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene® SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791;WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734. The antibodies can be used to identifyhomologs of the defensive agents of the invention.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1

Bioassay for Fungicidal Activity of Manduca sexta Hemolymph Polypeptides

After resolution by liquid chromatography (LC), the various pathogeninduced M. sexta polypeptide-containing fractions were assayed forfungicidal activity against the plant pathogens M. grisea, R. solani,and F. verticilloides. The LC fractions were first lyophilized in96-well microtitre plates. A suspension of 100 μl of M. grisea (or othernamed pathogen), at the standard fungal growth assay concentration (2500spores/ml), was added to the polypeptide containing microtitre platewells, and the plates sealed with Borden® Sealwrap™ wrap. The plateswere then placed at 28° C. in a dark chamber for 24 hours. Hyphal growthwas monitored using a dissecting microscope. The polypeptides containedin the wells that lacked hyphal growth, or that displayed reduced hyphalgrowth compared to control wells, were considered to possess fungicidalactivity. Hyphal growth was scored again, 48 hours post inoculation, fora final determination of fungicidal activity.

EXAMPLE 2

Induction of Antimicrobial Response in Manduca sexta

Fifth instar M. sexta larvae were injected intersegmentally with 20 μlof a highly concentrated suspension of M. grisea hyphae and sporespreviously scraped from an agar plate colony. The larvae were thenplaced on fresh diet and allowed to recover. After 24, 48, and 72 hours,hemolymph was collected from the larvae by clipping off a proleg usingfine surgical scissors over a sheet of Parafilm™ film. Approximately 1ml/insect can be collected in this way. The hemolymph was transferred toa 50 ml conical flask and placed on ice while the remaining larvae werebeing processed. Once all larvae have been processed, phenyl thiolureawas added to a final concentration of 20 mM. Aprotinin was also added tothe sample (final concentration 20 μg/ml). The samples were centrifuged(3000 rpm) for 5 minutes to pellet cells. The remaining supernatant(hemolymph) was subjected to solid-phase extraction using SupelcoDiscovery® DSC-18 solid-phase extraction columns. The columns arepreconditioned using 100% methanol, equilibrated using 100% Solvent A(5% acetonitrile, 0.1% TFA; 1 column volume) before the sample isloaded. After the hemolymph (supernatant) filters through, the column iswashed with Solvent A before eluting with one column volume of 60%Solvent B/40% Solvent A (Solvent B: 95% acetonitrile, 0.1% TFA). Thecollected eluent is frozen at −80 ° C. and lyophilized to dryness.Hemolymph samples are then resuspended in a small volume of water(usually 200-500 μL) and a BCA assay is done to determine proteinconcentration. Following the solid-phase fractionation step, thehemolymph samples are fractionated by HPLC and tested by bioassay.

Induction of M. sexta with B. bassiana and R. solani was performedsimilarly.

Corresponding pathogen (M. grisea; B. bassiana; R. solani) induced M.sexta cDNA libraries were constructed according to standard protocols.Briefly, total RNA was isolated from the fatbodies of pathogen inducedM. sexta. The mRNAs were isolated using an mRNA purification kit (BRL)according to the manufacture's instructions. The cDNA libraries wereconstructed using the ZAP-cDNA® synthesis kit and the pBluescript™phagemid (Stratagene®).

EXAMPLE 3

HPLC-Fractionation of Polypeptides from Maznaportha grisea InducedManduca sexta Hemolymph

Hemolymph from M. grisea induced M. sexta larvae (see Example 2) wasfractionated on HP-1100 HPLC, using a Vydac® C4 (4.6-250 mm) column(FIG. 3). A gradient system was used to elute bound proteins. Thegradient conditions are indicated below. Fractions were collected at oneminute intervals into a 96-well microtiter plate and were assayed forfungicidal activity against M. grisea (see Example 1).

This protocol was also followed for fractionation of polypeptides fromB. bassiana and R. solani induced M. sexta hemolymph. The bioassay forfungicidal activity (Example 1) was also conducted using the plantpathogens R. solani and F. verticilloides.

Gradient Conditions:

Solvent

-   Solvent A: 5% Acetonitrile, 0.1% TFA-   Solvent B: 95% Acetonitrile, 0.1% TFA    Flow Rate-   0.6 ml/min    Gradient-   0-60% B over 70 minutes

EXAMPLE 4

Microbore Purification of the Fungicidal Polypeptide Mag1

After fractionation by HPLC, those fractions from Example 3 possessingfungicidal activity (47-52 min fractions) were further separated bymicrobore LC (Michrom Bioresources, Inc., Auburn, Calif.) using a Vydac®C4 column (1-150 mm). The gradient conditions are indicated below. Thecolumn eluant was collected in such a manner as to best resolve thepeaks with the highest polypeptide content (FIG. 4). The elutedpolypeptides were assayed for fungicidal activity against M. grisea (SeeExample 1). The polypeptide fraction containing the greatest fungicidalactivity is indicated with an arrow.

Gradient Conditions:

Solvents

-   Solvent A: 5% Acetonitrile, 0.1% TFA-   Solvent B: 95% Acetonitrile, 0.1% TFA    Flow Rate-   50 μ/min    Gradient-   5-65% solvent B in 70 minutes

The polypeptide fraction containing the greatest fungicidal activity(indicated with an arrow in FIG. 4 was further resolved usingmicrobore-LC (Michrom Bioresources, Inc., Auburn, Calif.) on a Vydac®C18 (1-150 mm) column (FIG. 5). The gradient conditions follow. Againthe polypeptide-containing fractions were assayed for fungicidalactivity against M. grisea (See Example 1). (The resulting purifiedpolypeptide was designated Mag1.)

Gradient Conditions

Solvents

-   Solvent A: 5% Acetonitrile, 0.1% HFBA-   Solvent B: 95% Acetonitrile, 0.1% HFBA    Flow Rate-   50 μl/min    Gradient-   5-65% solvent B in 70 minutes

This protocol was also followed for microbore purification of fungicidalpolypeptides identified in B. bassiana and R. solani induced M. sextahemolymph. The bioassay for fungicidal activity (Example 1) was alsoconducted using the plant pathogens R. solani and F. verticilloides.

EXAMPLE 5

Molecular Weight Determination of Mag1

The molecular weight of the isolated Mag1 polypeptide from Example 4 wasdetermined using Liquid Chromatography-Mass Spectrophotometry (LC-MS).The molecular mass of Mag1 was determined using electrospray massspectrometry on a Micromass® platform LCZ mass spectrometer (Micromass,Manchester, UK). Microbore LC (Michrom Bioresources, Auburn, Calif.)delivered the protein and mobile phase (acetonitrile/water) using areversed phase column. Spectra were obtained in positive ion mode usinga capillary voltage of 3.5 kV, a cone voltage of 45V, and a sourcetemperature of 90° C. Spectra scanned over a range of 600-3000 at a rateof 3.5 s/scan. Molecular masses were determined using the maximumentropy deconvolution algorithm (MaxEnt) to transform the m/z range600-3000 to give a true mass scale spectrum. Mass calibration wasperformed using horse heart myoglobin.

A similar protocol was performed for the other polypeptides of theinvention.

EXAMPLE 6

Lys-C Endoproteinase Digestion of Mag1

Sequencing grade lyophilized endoproteinase Lys-C (Boehringer Mannheim)was reconstituted in 50 μl redistilled water resulting in a bufferconcentration of 50 mM Tricine pH 8.0, 10 mM EDTA, and 0.5 mg/mlraffinose. The Mag1 polypeptide from Example 4 was dissolved indigestion buffer (25 mM Tris HCl pH 8.5, 1 nM EDTA) to a ratio of 1:50Lys-C to Mag1 polypeptide by weight. The reaction was allowed to proceedfor 20 hours at 37° C. The digested polypeptide was fractionated using aC4 column on a microbore-HPLC with a gradient of 5-65% acetonitrile in0.1% TFA over 70 minutes at a flow rate of 50 μl/min (FIG. 6). Fourisolated fragments were collected and submitted for N-terminal sequenceanalysis.

A similar protocol was followed for digestion of the other fungicidalpolypeptides of the invention.

EXAMPLE 7

N-Terminal Amino Acid Sequence Determination of Mag1 PolypeptideFragments

The N-termini of the isolated Mag1 fragments from Example 6 weresequenced on an ABI Procise® 494 Protein Sequencer, consisting of achemistry workstation, a PTH analysis system, computer control and anautomated sequence calling software. Standard protocols were used to runthe system and determine the sequences (see FIG. 7).

The N-terminal amino acid sequences of isolated fragments of the otherpolypeptides of the invention were determined similarly.

N-terminal peptide sequence is critical in determining the exact orprecise processing site for the conversion of the pro-peptide into themature and active form of the protein (as in this example, Mag1). Thisis in particular important for secretory proteins.

C-terminal peptide sequence was deduced from both the molecular weightgenerated by LC-MS of the active protein and the predicted molecularweight of the same encoded polypeptide based of the identified cDNAsequence (in Example 8).

By knowing the precise termini of the mature protein, one can design andconstruct DNA molecules that encode the entire active mature protein forexpression in plants. When necessary, additional plant specificcontrolling elements and targeting sequences can be tailored andincorporated in the gene design in order to enhance and target theexpression of the mature polypeptide in plants.

To ensure the original specificity and functionality of the e.g. Mag1protein retained in the plant, the expression of the active mature formof the protein in the plant is essential.

EXAMPLE 8

Isolation of the cDNA Clone Encoding Mag1

Fat bodies were harvested directly into liquid nitrogen beforeprocessing. Total RNA from fatbodies of challenged Manduca sexta wasprepared by pulverizing the tissue with a mortar and pestle in liquidnitrogen and lysing cells in the presence of TRIzol® (Life Technologies,Inc.) according to the manufacturer's protocol. PolyA(+) RNA wasoligo(dT)-cellulose affinity purified from total RNA using the mRNAPurification Kit (Amersham Pharmacia Biotech) following themanufacturer's protocol in preparation for cDNA library construction.First strand cDNA synthesis using Superscript II (Life Technologies,Inc.) and subsequent second strand synthesis, linker addition, anddirectional cloning into restriction sites of pBlueScript™ SK+ plasmid(Stratagene®) was performed according to the instructions provided withthe Stratagene® cDNA kit (Stratagene®). cDNA was purified using a cDNAcolumn (Life Technologies, Inc.) immediately prior to ligation into thevector.

Sequencing of the cDNA library clones was performed using the ABI PRISM®Big Dye Terminator Cycle Sequencing Ready Kit with FS AmpliTaq DNApolymerase (Perkin Elmer™) and analyzed on an ABI Model 373 AutomatedDNA Sequencer. The Mag1 gene sequence was identified by sequencing about2000 clones of the cDNA library prepared from mRNA derived from thefatbodies of challenged M. sexta. Amino acid sequences derived fromamino termini of the complete peptide or proteolytic cleavage productswere used to compare to the corresponding cDNA clone sequence librarytranslated in the six possible frames. Sequences containing 100%identity to the N-terminal amino acid sequences were fully translatedand their predicted MW compared to the MW of the purified Mag1 protein.Sequences with comparable MWs were identified as probably encoding Mag1.

EXAMPLE 9

Isolation of the cDNA Clone Encoding a Polypeptide of Interest

The N-terminal amino acid sequence tags of a polypeptide of interest areused to identify cDNA clones encoding the polypeptide. Degenerateoligonucleotides encoding the amino acid sequence tags of thepolypeptide are used as probes to detect cDNA's encoding the polypeptidein a pathogen induced M. sexta cDNA library (see Example 2). In thismanner a full-length cDNA encoding the polypeptide of interest isisolated and sequenced. Complete sequencing of the identified cDNA cloneis performed to confirm that it encodes the purified polypeptide.Confirmation is provided by the predicted molecular weight of the cDNAencoded polypeptide being the same as the molecular weight of thepolypeptide generated by LC-MS.

EXAMPLE 10

Construction of Recombinant Baculovirus Expressing FungicidalPolypeptides

The nucleotide sequences encoding the polypeptides of the invention maybe introduced into the baculovirus genome itself. For this purpose thenucleotide sequences may be placed under the control of the polyhedrinpromoter, the IE1 promoter, or any other one of the baculoviruspromoters. The cDNA, together with appropriate leader sequences is theninserted into a baculovirus transfer vector using standard molecularcloning techniques. Following transformation of E. coli DH5α, isolatedcolonies are chosen and plasmid DNA is prepared and is analyzed byrestriction enzyme analysis. Colonies containing the appropriatefragment are isolated, propagated, and plasmid DNA is prepared forcotransfection.

EXAMPLE 11

Expression of Fungicidal Polypeptides in Insect Cells

The polypeptides of the invention may be expressed in insect cells. Forthis purpose the Spodoptera frugiperda cells (Sf-9 or Sf-21) arepropagated in ExCell® 401 media (JRH Biosciences™, Lenexa, Kans.), or asimilar media, supplemented with 3.0% fetal bovine serum. Lipofectin®(50 μL at 0.1 mg/mL, Gibco BRL) is added to a 50 μL aliquot of thetransfer vector containing the antimicrobial nucleotide sequences (500ng) and linearized polyhedrin-negative AcNPV (2.5 μg, Baculogold® viralDNA, Pharmingen®, San Diego, Calif.). Sf-9 cells (approximate 50%monolayer) are co-transfected with the viral DNA/transfer vectorsolution. The supernatant fluid from the co-transfection experiment iscollected at 5 days post-transfection and recombinant viruses areisolated employing standard plaque purification protocols, wherein onlypolyhedrin-positive plaques are selected (O'Reilly et al. (1992),Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman andCompany, N.Y.). Sf-9 cells in 35 mm petri dishes (50% monolayer) areinoculated with 100 μL of a serial dilution of the viral suspension, andsupernatant fluids are collected at 5 days post infection. In order toprepare larger quantities of virus for characterization, thesesupernatant fluids are used to inoculate larger tissue cultures forlarge scale propagation of recombinant viruses. Expression of theencoded fungicidal polypeptide by the recombinant baculovirus can beconfirmed using a bioassay (such as described in Example 4), LC-MS, orantibodies.

EXAMPLE 12

Expression of Fungicidal Peptides in Pichia

The nucleotide sequences encoding the polypeptides of the invention maybe expressed in Pichia under constitutive or inducible promoter controland targeted to remain intracellular or to be secreted into the media.The nucleotide sequences are cloned into a Pichia expression vectorusing standard molecular techniques. Transformation of Pichia strains(e.g. X-33, GS 115, SMD1168, KM71, etc. —Invitrogen™, Carlsbad, Calif.)involves linearization of the construct and introduction of the DNA intotransformation competent Pichia cells by chemical means or byelectroporation according to standard protocols. Transformants areselected by either resistance to Zeocin or blasticidin or by theirability to grow on histidine-deficient medium. Small scale expressiontests are performed on selected transformants to identify highexpressors of the polypeptides of the invention for additional scale up.In an inducible system, such as when the peptide is under control of theAOX1 promoter, transformants are grown in media with glycerol as acarbon source and induced by growth in media containing methanol insteadof glycerol. Continuous induction over a period of 24-120 hrs isachieved by addition of methanol (0.5% final conc.) every 24 hr.Functional expression of the polypeptide is confirmed by LC-MSanalysis/purification and bioassay.

EXAMPLE 13

Expression of Fungicidal Polypeptides in Bacteria

The nucleotide sequences encoding the polypeptides of the invention maybe expressed in bacteria and the peptides targeted for intracellular orextracellular expression. The cDNA's may be cloned into a suitablebacterial expression vector (e.g. pET vectors (Novagen®, Madison, Wis.)under constitutive or inducible promoter control using standardmolecular cloning techniques. The plasmid containing the gene ofinterest is introduced into transformation competent bacteria cellsusing standard protocols for chemical transformation or electroporationand the transformants are selected using antibiotic resistance. Inaddition to traditional E. coli strains commonly used fortransformation, mutant strains such as Origami™ (Novagen®, Madison,Wis.) that are permissive for disulfide bond formation can be used,especially with cysteine-rich peptides to express functional peptides.Inducible systems such as E. coli strains bearing the T7 RNA polymerasegene (lambda- DE3 lysogen) can be used in which expression of the geneof interest under a T7 promoter is induced by addition of IPTG forvariable periods of time. Expression and activity of the polypeptidesare confirmed by LC-MS and bioassays.

EXAMPLE 14

Transformation of Rice Embryogenic Callus by Bombardment andRegeneration of Transgenic Plants

Embryogenic callus cultures derived from the scutellum of germinatingseeds serve as the source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is then transferred toCM media (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu etal., 1985, Sci. Sinica 18:659-668). Callus cultures are maintained on CMby routine sub-culture at two week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman® #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C/ for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hoursin the dark. The petri dish lids are then left ajar for 20-45 minutes ina sterile hood to allow moisture on tissue to dissipate.

Circular plasmid DNA from two different plasmids one containing theselectable marker for rice transformation and one containing thenucleotide of the invention, are co-precipitated onto the surface ofgold particles. To accomplish this, a total of 10 μg of DNA at a 2:1ratio of trait: selectable marker DNAs is added to a 50 μl aliquot ofgold particles resuspended at a concentration of 60 mg/ml. Calciumchloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a 0.1 Msolution) are then added to the gold-DNA suspension as the tube isvortexing for 3 minutes. The gold particles are centrifuged in amicrofuge for 1 sec and the supernatant removed. The gold particles arethen washed twice with 1 ml of absolute ethanol and then resuspended in50 μl of absolute ethanol and sonicated (bath sonicator) for one secondto disperse the gold particles. The gold suspension is incubated at −70°C. for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six microliters of the DNA-coated gold particles are thenloaded onto mylar macrocarrier disks and the ethanol is allowed toevaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 p.s.i.The tissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Five to seven plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipette. Threemilliliter aliquots of the callus suspension are plated onto fresh SMmedia and the plates incubated in the dark for 4 weeks at 27-28° C.After 4 weeks, transgenic callus events are identified, transferred tofresh SM plates and grown for an additional 2 weeks in the dark at27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% Gelrite™ gelling agent+ 50 ppmhyg B) for 2 weeks in the dark at 25° C. After 2 weeks the callus istransferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3%sucrose, 0.4% Gelrite™ gelling agent+ 50 ppm hyg B) and placed undercool white light (˜40 μEm⁻²S⁻¹) with a 12 hr photoperiod at 25° C. and30-40% humidity. After 2-4 weeks in the light, callus generally beginsto organize, and form shoots. Shoots are removed from surroundingcallus/media and gently transferred to RM3 media (½×MS salts, Nitsch andNitsch vitamins, 1% sucrose +50 ppm hygromycin B) in phytatrays (Sigma®Chemical Co., St. Louis, Mo.) and incubation is continued using the sameconditions as described in the previous step.

Plants are transferred from RM3 to 4″ pots containing Metro mix 350after 2-3 weeks, when sufficient root and shoot growth has occurred.Plants are grown using a 12 hr/12 hr light/dark cycle using˜30/18° C.day/night temperature regimen.

EXAMPLE 15

Transformation of Maize by Particle Bombardment and Regeneration ofTransgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing a nucleotide sequence of the invention operablylinked to a ubiquitin promoter and the selectable marker gene PAT(Wohlleben et al. (1988) Gene 70:25-37), which confers resistance to theherbicide Bialaphos. Alternatively, the selectable marker gene isprovided on a separate plasmid. Transformation is performed as follows.Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Cloro™ bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising the nucleotide sequence of the inventionoperably linked to a ubiquitin promoter is made. This plasmid DNA plusplasmid DNA containing a PAT selectable marker is precipitated onto 1.1μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

-   -   100 μl prepared tungsten particles in water    -   10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μl 2.5M CaCl₂    -   10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the nucleotidesequence encoding the fungicidal polypeptide of the invention, or forthe presence of the fungicidal polypeptide by immunological methods, orfor fungicidal activity by assays known in the art, described supraherein.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (Sigma®C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline(brought to volume with D-I H₂0 following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite™ gelling agent (added after bringing to volumewith D-I H₂0); and 8.5 mg/l silver nitrate (added after sterilizing themedium and cooling to room temperature). Selection medium (560R)comprises 4.0 g/l N6 basal salts (Sigma®C-1416), 1.0 ml/l Eriksson'sVitamin Mix (1000× Sigma®-1511), 0.5 mg/l thiamine HCl, 30.0 g/lsucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H₂0 followingadjustment to pH 5.8 with KOH); 3.0 g/l Gelrite™ gelling agent (addedafter bringing to volume with D-I H₂0); and 0.85 mg/l silver nitrate and3.0 mg/l bialaphos (both added after sterilizing the medium and coolingto room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts(Gibco®1117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/lglycine brought to volume with polished D-I H₂0) (Murashige and Skoog(1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin,60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volumewith polished D-I H₂0 after adjusting to pH 5.6); 3.0 g/l Gelrite™gelling agent (added after bringing to volume with D-I H₂0); and 1.0mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizingthe medium and cooling to 60° C.). Hormone-free medium (272V) comprises4.3 g/l MS salts (Gibco(® 11117-074), 5.0 ml/l MS vitamins stocksolution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/lpyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-IH₂0), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume withpolished D-I H₂0 after adjusting pH to 5.6); and 6 g/l bacto-agar (addedafter bringing to volume with polished D-I H₂0), sterilized and cooledto 60° C.

EXAMPLE 16

Agrobacterium-Mediated Transformation of Maize and Regeneration ofTransgenic Plants

For Agrobacterium-mediated transformation of maize with aplant-optimized nucleotide sequence of the invention, preferably themethod of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patentpublication WO98/32326; the contents of which are hereby incorporated byreference). Briefly, immature embryos are isolated from maize and theembryos contacted with a suspension of Agrobacterium, where the bacteriaare capable of transferring the plant-optimized nucleotide sequence ofthe invention to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryosare preferably immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Preferably theimmature embryos are cultured on solid medium following the infectionstep. Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

EXAMPLE 17

Transformation of Soybean Embryos and Regeneration of Transgenic Plants

Soybean embryos are bombarded with a plasmid containing a nucleotidesequence of the invention operably linked to a ubiquitin promoter asfollows. To induce somatic embryos, cotyledons, 3-5 mm in lengthdissected from surface-sterilized, immature seeds of the soybeancultivar A2872, are cultured in the light or dark at 26° C. on anappropriate agar medium for six to ten weeks. Somatic embryos producingsecondary embryos are then excised and placed into a suitable liquidmedium. After repeated selection for clusters of somatic embryos thatmultiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can maintained in 35 ml liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 ml of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont™ Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the nucleotide sequenceof the invention operably linked to the ubiquitin promoter can beisolated as a restriction fragment. This fragment can then be insertedinto a unique restriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 18

Transformation of Sunflower Meristem Tissue and Regeneration ofTransgenic Plants

Sunflower meristem tissues are transformed with an expression cassettecontaining the nucleotide sequence of the invention operably linked to aubiquitin promoter as follows (see also European Patent Number EP 0486233, herein incorporated by reference, and Malone-Schoneberg et al.(1994) Plant Science 103:199-207). Mature sunflower seed (Helianthusannuus L.) are dehulled using a single wheat-head thresher. Seeds aresurface sterilized for 30 minutes in a 20% Clorox™ bleach solution withthe addition of two drops of Tween™ 20 per 50 ml of solution. The seedsare rinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer et al. (Schrammeijer et al.(1990)Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled water for 60minutes following the surface sterilization procedure. The cotyledons ofeach seed are then broken off, producing a clean fracture at the planeof the embryonic axis. Following excision of the root tip, the explantsare bisected longitudinally between the primordial leaves. The twohalves are placed, cut surface up, on GBA medium consisting of Murashigeand Skoog mineral elements (Murashige et al. (1962) Physiol. Plant., 15:473-497), Shepard's vitamin additions (Shepard (1980) in EmergentTechniques for the Genetic Improvement of Crops (University of MinnesotaPress, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/l sucrose, 0.5mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-acetic acid (IAA),0.1 mg/l gibberellic acid (GA₃), pH 5.6, and 8 g/l Phytagar™ agar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney et al. (1992) Plant Mol. Biol. 18:301-313). Thirty to forty explants are placed in a circle at the centerof a 60×20 mm plate for this treatment. Approximately 4.7 mg of 1.8 mmtungsten microprojectiles are resuspended in 25 ml of sterile TE buffer(10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used perbombardment. Each plate is bombarded twice through a 150 mm nytex screenplaced 2 cm above the samples in a PDS 1000® particle accelerationdevice.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the nucleotide sequence of theinvention operably linked to the ubiquitin promoter is introduced intoAgrobacterium strain EHA105 via freeze-thawing as described by Holsterset al. (1978) Mol. Gen. Genet. 163:181-187. This plasmid furthercomprises a kanamycin selectable marker gene (i.e., nptII). Bacteria forplant transformation experiments are grown overnight (28° C. and 100 RPMcontinuous agitation) in liquid YEP medium (10 gm/l yeast extract, 10gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the appropriateantibiotics required for bacterial strain and binary plasmidmaintenance. The suspension is used when it reaches an OD₆₀₀ of about0.4 to 0.8. The Agrobacterium cells are pelleted and resuspended at afinal OD₆₀₀ of 0.5 in an inoculation medium comprised of 12.5 mM MES pH5.7, 1 gm/l NH₄Cl, and 0.3 gm/l MgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for expression of the nucleotidesequence encoding the fungicidal polypeptide of the invention, thepresence of the fungicidal polypeptide by immunological methods, or forfungicidal activity by assays known in the art, described supra herein.

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%Gelrite™ gelling agent, pH 5.6) and grown under conditions described forexplant culture. The upper portion of the seedling is removed, a 1 cmvertical slice is made in the hypocotyl, and the transformed shootinserted into the cut. The entire area is wrapped with Parafilm™ film tosecure the shoot. Grafted plants can be transferred to soil followingone week of in vitro culture. Grafts in soil are maintained under highhumidity conditions followed by a slow acclimatization to the greenhouseenvironment. Transformed sectors of T₀ plants (parental generation)maturing in the greenhouse are identified by NPTII ELISA and/or by thefungicidal activity analysis of leaf extracts while transgenic seedsharvested from NPTII-positive To plants are identified by fungicidalactivity analysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Thismethod is generally used in cases where the nucleotide sequences of thepresent invention are operably linked to constitutive or induciblepromoters. Seeds are dehulled and surface-sterilized for 20 minutes in a20% Clorox™ bleach solution with the addition of two to three drops ofTween™ 20 per 100 ml of solution, then rinsed three times with distilledwater. Sterilized seeds are imbibed in the dark at 26° C. for 20 hourson filter paper moistened with water. The cotyledons and root radicalare removed, and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagar™agar at pH 5.6) for 24 hours under the dark. The primary leaves areremoved to expose the apical meristem, around 40 explants are placedwith the apical dome facing upward in a 2 cm circle in the center of374M (GBA medium with 1.2% Phytagar™ agar), and then cultured on themedium for 24 hours in the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bactopeptone, and 5 g/l NaCl, pH 7.0) in the presence of50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄Cl and 0.3 g/l MgSO₄at pH 5.7) to reach a final concentration of 4.0 at OD 600.Particle-bombarded explants are transferred to GBA medium (374E), and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for the expression of the nucleotide sequence of the inventionor the presence of the encoded polypeptide of the invention byimmunological methods or fungicidal activity, or the like. Afterpositive explants are identified, those shoots that fail to exhibitfungicidal activity are discarded, and every positive explant issubdivided into nodal explants. One nodal explant contains at least onepotential node. The nodal segments are cultured on GBA medium for threeto four days to promote the formation of auxiliary buds from each node.Then they are transferred to 374C medium and allowed to develop for anadditional four weeks. Developing buds are separated and cultured for anadditional four weeks on 374C medium. Pooled leaf samples from eachnewly recovered shoot are screened again by the appropriate proteinactivity assay. At this time, the positive shoots recovered from asingle node will generally have been enriched in the transgenic sectordetected in the initial assay prior to nodal culture.

Recovered shoots positive for a fungicidal polypeptide of the inventionare grafted to Pioneer® hybrid 6440 in vitro-grown sunflower seedlingrootstock. The rootstocks are prepared in the following manner. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Clorox™bleach solution with the addition of two to three drops of Tween™ 20 per100 ml of solution, and are rinsed three times with distilled water. Thesterilized seeds are germinated on the filter moistened with water forthree days, then they are transferred into 48 medium (half-strength MSsalt, 0.5% sucrose, 0.3% Gelrite™ gelling agent pH 5.0) and grown at 26°C. under the dark for three days, then incubated at 16-hour-day cultureconditions. The upper portion of selected seedling is removed, avertical slice is made in each hypocotyl, and a transformed shoot isinserted into a V-cut. The cut area is wrapped with Parafilm™ film.After one week of culture on the medium, grafted plants are transferredto soil. In the first two weeks, they are maintained under high humidityconditions to acclimatize to a greenhouse environment.

EXAMPLE 19

Preparation of Antibodies.

Standard methods for the production of antibodies were used such asthose described in Harlow and Lane (1988) Antibodies: A LaboratoryManual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory),incorporated herein in its entirety by reference. Specifically,antibodies for polypeptides of the invention were produced by injectingfemale New Zealand white rabbits (Bethyl Laboratory, Montgomery, Tex.)six times with 100 micrograms of denatured purified polypeptide.

Animals were then bled at two week intervals. The antibodies werepurified by affinity-chromatography with Affigel 15 (Bio-Rad®Laboratories, Inc., Hercules, Calif.)-immobilized antigen as describedby Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor, N.Y. The affinity column was prepared with purified polypeptideessentially as recommended by the manufacturer. Immune detection ofantigens on PVDF blots was carried out following the protocol of Meyeret al. (1988) J. Cell. Biol. 107:163; incorporated herein in itsentirety by reference, using the ECL kit from Amersham™ Corporation(Arlington Heights, Ill.).

EXAMPLE 20

Construction of Fus1 Transformation Vector

A synthetic version of the Fus1 gene corresponding to the mature Fus1peptide was constructed with a codon-bias representative of Manducasexta (SEQ ID NO:120 and SEQ ID NO:122). The codon preference selectedfor Fus1 was derived from the Kazusa codon usage database (availablefrom www.Kazusa.or.jp/codon/). The BAA signal sequence was added to Fus1to facilitate export of out of the cell and into the intercellular space(Rahmatullah R J et al. (1989) Plant Mol. Biol. 12(1):119-121). TheBAA-Fus1 amino acid sequence is set forth in SEQ ID NO:121 and SEQ IDNO:123. Strong constitutive promoters were chosen to express Fus1 intissues susceptible to F. verticilloides. BAA-Fus1 (SEQ ID NO:120) wassubsequently subcloned into the corresponding sites of vectorscontaining either the maize ubiquitin promoter:ubi-intron or the maizeh2B promoter:ubi-intron (U.S. Pat. No. 6,177,611, herein incorporated byreference). BAA-Fus 1 was placed behind the indicated promoter with a 3′sequence corresponding to the pinII terminator. This cassette is flankedby non-compatible restriction enzyme sites designed to directionallyclone the cassette into a binary plasmid containing the selectablemarker gene cassette 35S-PAT-35S. The restriction enzyme sites were usedto subclone the promoter/intron:BAA-Fus1 :pinII ter cassette into abinary plasmid for corn transformation.

EXAMPLE 21

Construction of Fus2 Transformation Vectors

A synthetic version of Fus2 operably linked to a modified barley alphaamylase (BAA) signal peptide was constructed with a codon-biasrepresentative of Streptomyces coelicolor (SEQ ID NO:124 and SEQ IDNO:126). S. coelicolor codon usage was chosen because of its overallsimilarity to the codon usage observed in plants. The codon preferenceselected for Fus2 was derived from the Kazusa codon usage database(available from www.Kazusa.or.jp/codon/). See also Tables 1 and 2. TheBAA signal sequence was added to Fus2 to facilitate export of Fus2 outof the cell and into the intercellular space. Modifications to the 3′end of the signal peptide were made to achieve correct signal peptidecleavage as predicted by the SIGNALP (Version 1.1) program (Center forBiological Sequence Analysis, Technical University of Denmark). TheBAA-Fus2 amino acid sequence is set forth in SEQ ID NO:125 and SEQ IDNO:127. The synthetic gene was constructed using a series of overlappingcomplementary oligonucleotides that were annealed together, Klenowtreated to repair the gaps, and PCR amplified using primerscorresponding to 5′ and 3′ ends of the synthetic gene. Restrictionenzyme sites were incorporated into the PCR primers to facilitate genecloning. The PCR product was TOPO cloned into pCR2. 1 (Invitrogen™) andsequence verified. A restriction enzyme fragment containing BAA-Fus2 wassubsequently subcloned into the corresponding sites of vectorscontaining either the maize ubiquitin promoter: ubi-intron or the maizeh2B promoter:ubi-intron. The vectors contained a 3′ sequencecorresponding to the pinII terminator. The BAA-Fus2 fragment was clonedbetween the indicated promoter and the pinII terminator. Strongconstitutive promoters were chosen to express Fus2 in tissuessusceptible to F. verticilloides. The promoter/intron:BAA-Fus2:pinII tercassette is flanked by non-compatible restriction enzyme sites designedto directionally clone the cassette into a binary plasmid containing aselectable marker. The restriction enzyme sites were used to subclonethe promoter/intron:BAA-Fus2:pinII ter cassette into a binary plasmidfor corn transformation. TABLE 1 Streptomyces coelicolor A3(2) [gbbct]:6257 CDS's (2043281 codons) fields: [triplet] [frequency: per thousand]([number]) UUU  0.4 (863) UCU  0.6 (1266) UAU  1.0 (1962) UGU  0.7(1448) UUC 26.0 (53065) UCC 20.2 (41262) UAC 19.5 (39789) UGC  7.0(14341) UUA  0.1 (128) UCA  1.0 (2137) UAA  0.1 (290) UGA  2.4 (4878)UUG  2.4 (4935) UCG 13.8 (28229) UAG  0.5 (1089) UGG 15.1 (30770) CUU 1.5 (3129) CCU  1.5 (2995) CAU  1.6 (3366) CGU  5.5 (11183) CUC 36.6(74736) CCC 25.4 (51951) CAC 21.5 (44018) CGC 39.1 (79956) CUA  0.3(657) CCA  1.3 (2633) CAA  1.3 (2593) CGA  2.5 (5124) CUG 61.3 (125241)CCG 33.6 (68652) CAG 25.1 (51248) CGG 32.0 (65332) AUU  0.6 (1228) ACU 1.1 (2347) AAU  0.7 (1436) AGU  1.5 (3030) AUC 27.6 (56340) ACC 39.6(80826) AAC 16.2 (33191) AGC 12.3 (25187) AUA  0.7 (1367) ACA  1.6(3194) AAA  1.0 (2041) AGA  0.8 (1574) AUG 15.8 (32271) ACG 18.9 (38697)AAG 19.7 (40293) AGG  3.7 (7488) GUU  1.4 (2905) GCU  2.9 (5908) GAU 2.9 (6024) GGU  9.3 (18920) GUC 47.2 (96460) GCC 78.6 (160548) GAC 58.0(118595) GGC 61.4 (125467) GUA  2.7 (5416) GCA  5.3 (10890) GAA  8.5(17445) GGA  7.1 (14608) GUG 35.3 (72144) GCG 49.8 (101831) GAG 48.5(99056) GGG 18.2 (37288)Coding GC 72.38% 1st letter GC 72.74% 2nd letter GC 51.39% 3rd letter GC93.00%

TABLE 2 Streptomyces coelicolor [gbbct]: 2110 CDS's (646333 codons)fields: [triplet] [frequency: per thousand] ([number]) UUU  0.5 (329)UCU  0.8 (496) UAU  1.0 (676) UGU  0.8 (517) UUC 25.7 (16596) UCC 20.1(12971) UAC 19.4 (12521) UGC  7.3 (4734) UUA  0.1 (49) UCA  1.2 (797)UAA  0.2 (105) UGA  2.6 (1650) UUG  2.6 (1696) UCG 13.5 (8729) UAG  0.5(355) UGG 15.2 (9813) CUU  1.9 (1228) CCU  1.8 (1178) CAU  1.9 (1251)CGU  5.6 (3602) CUC 36.2 (23411) CCC 25.4 (16419) CAC 22.6 (14594) CGC39.2 (25310) CUA  0.5 (304) CCA  1.6 (1018) CAA  1.7 (1076) CGA  2.9(1885) CUG 59.3 (38346) CCG 32.7 (21145) CAG 25.8 (16671) CGG 31.5(20333) AUU  0.8 (497) ACU  1.4 (925) AAU  0.8 (515) AGU  1.6 (1023) AUC27.8 (17997) ACC 39.9 (25804) AAC 16.2 (10447) AGC 12.7 (8194) AUA  0.7(444) ACA  1.9 (1245) AAA  1.3 (829) AGA  0.8 (537) AUG 16.1 (10392) ACG19.1 (12377) AAG 19.8 (12795) AGG  3.8 (2441) GUU  1.7 (1086) GCU  3.8(2429) GAU  3.5 (2251) GGU  9.1 (5867) GUC 46.3 (29904) GCC 77.5 (50098)GAC 58.2 (37624) GGC 58.8 (38034) GUA  2.7 (1767) GCA  6.7 (4302) GAA 9.6 (6215) GGA  7.3 (4689) GUG 33.9 (21929) GCG 48.6 (31399) GAG 47.9(30970) GGG 17.8 (11502)Coding GC 71.94% 1st letter GC 72.38% 2nd letter GC 51.28% 3rd letter GC92.14%

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated nucleic acid molecule comprising a nucleotide sequencethat encodes a polypeptide having defensive activity, wherein saidnucleotide sequence is selected from the group consisting of: (a) thenucleotide sequence which is the coding sequence set forth in SEQ IDNO:1; (b) a nucleotide sequence comprising at least 50 contiguousnucleotides of the sequence set forth in SEQ ID NO:1; (c) a nucleotidesequence that encodes the polypeptide set forth in SEQ ID NO:2; (d) anucleotide sequence that encodes a polypeptide having at least 90%sequence identity to the amino acid sequence set forth in SEQ ID NO:2;(e) a nucleotide sequence having at least 90% sequence identity to anucleotide sequence that encodes the amino acid sequence set forth inSEQ ID NO:2;and (f) a nucleotide sequence consisting of the complementof any one of the nucleotide sequences in (a), (b), (c), (d), or (e). 2.A vector comprising the nucleic acid molecule of claim
 1. 3. A host cellcomprising a construct comprising a promoter operably linked to thenucleic acid molecule of claim 1 (a), (b), (c), (d), or (e).
 4. The hostcell of claim 3, wherein the host cell is from an organism selected fromthe group consisting of fungi, yeast, and plants.
 5. A virus comprisingthe isolated nucleic acid molecule of claim 1 (a), (b), (c), (d), or(e).
 6. An expression cassette comprising the nucleic acid molecule ofclaim 1 operably linked to a promoter that drives expression in a plantcell.
 7. The expression cassette of claim 6, wherein said promoter isselected from the group consisting of inducible promoters andtissue-preferred promoters.
 8. The expression cassette of claim 7,wherein said promoter is a pathogen-inducible promoter.
 9. A transformedplant comprising in its genome at least one stably incorporatedexpression cassette comprising a nucleotide sequence operably linked toa promoter that drives expression in said plant cell, wherein saidnucleotide sequence encodes a polypeptide that has defensive activityand wherein said nucleotide sequence is selected from the groupconsisting of: (a) the nucleotide sequence which is the coding sequenceset forth in SEQ ID NO:1; (b) a nucleotide sequence comprising at least50 contiguous nucleotides of the sequence set forth in SEQ ID NO:1; (c)a nucleotide sequence that encodes the polypeptide set forth in SEQ IDNO:2; (d) a nucleotide sequence that encodes a polypeptide having atleast 90% sequence identity to the amino acid sequence set forth in SEQID NO:2; and (e) a nucleotide sequence having at least 90% sequenceidentity to a nucleotide sequence that encodes the amino acid sequenceset forth in SEQ ID NO:2.
 10. The transformed plant of claim 9, whereinsaid promoter is selected from the group consisting of induciblepromoters and tissue-preferred promoters.
 11. The transformed plant ofclaim 10, wherein said promoter is a pathogen-inducible promoter. 12.The transformed plant of claim 9, wherein said plant is selected fromthe group consisting of rice, corn, alfalfa, sunflower, Brassica,soybean, cotton, safflower, peanut, sorghum, wheat, millet, and tobacco.13. A transformed seed of the plant of claim 9, wherein said transformedseed comprises said nucleotide sequence.
 14. A method for enhancingdisease resistance of a plant to a fungal pathogen, said methodcomprising: (a) transforming a plant cell with at least one stablyincorporated expression cassette comprising a nucleotide sequenceoperably linked to a promoter that drives expression in a cell of saidplant, wherein said nucleotide sequence encodes a polypeptide that hasdefensive activity and wherein said polypeptide is selected from thegroup consisting of: (i) the nucleotide sequence which is the codingsequence set forth in SEQ ID NO:1; (ii) a nucleotide sequence comprisingat least 50 contiguous nucleotides of the sequence set forth in SEQ IDNO:1; (iii) a nucleotide sequence that encodes the polypeptide set forthin SEQ ID NO:2; (iv) a nucleotide sequence that encodes a polypeptidehaving at least 90% sequence identity to the amino acid sequence setforth in SEQ ID NO:2; (v) a nucleotide sequence having at least 90%sequence identity to a nucleotide sequence that encodes the amino acidsequence set forth in SEQ ID NO:2; and (vi) a nucleotide sequenceconsisting of the complement of any one of the nucleotide sequences in(a), (b), (c), (d), or (e); and (b) regenerating a transformed plantfrom said plant cell, wherein the level of resistance to said fungalpathogen in said plant is increased in comparison to a plant that doesnot comprise said expression cassette.
 15. The method of claim 14,wherein said plant is selected from the group consisting of rice, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, and tobacco.
 16. The method of claim 15, whereinsaid plant possesses enhanced resistance to Magnaportha grisea,Rhizoctonia solani, or Fusarium verticilloides.